Trialkyl cationic lipids and methods of use thereof

ABSTRACT

The present invention provides compositions and methods for the delivery of therapeutic agents to cells. In particular, these include novel, trialkyl, cationic lipids and nucleic acid-lipid particles that provide efficient encapsulation of nucleic acids and efficient delivery of the encapsulated nucleic acid to cells in vivo. The compositions of the present invention are highly potent, thereby allowing effective knock-down of a specific target protein at relatively low doses.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/380,536, which is a 35 U.S.C. §371 application of InternationalApplication No. PCT/US2013/027469, filed Feb. 22, 2013, which claims thebenefit of U.S. Provisional Application Ser. No. 61/602,990, filed onFeb. 24, 2012. The entire content of the applications referenced aboveare hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates to novel trialkyl cationic lipids, lipidparticles comprising one or more of the trialkyl cationic lipids,methods of making the lipid particles, and methods of delivering and/oradministering the lipid particles (e.g., for treating disease inmammals).

II. Description of the Related Art

Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA),microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, andimmune-stimulating nucleic acids. These nucleic acids act via a varietyof mechanisms. In the case of interfering RNA molecules such as siRNAand miRNA, these nucleic acids can down-regulate intracellular levels ofspecific proteins through a process termed RNA interference (RNAi).Following introduction of interfering RNA into the cell cytoplasm, thesedouble-stranded RNA constructs can bind to a protein termed RISC. Thesense strand of the interfering RNA is displaced from the RISC complex,providing a template within RISC that can recognize and bind mRNA with acomplementary sequence to that of the bound interfering RNA. Havingbound the complementary mRNA, the RISC complex cleaves the mRNA andreleases the cleaved strands. RNAi can provide down-regulation ofspecific proteins by targeting specific destruction of the correspondingmRNA that encodes for protein synthesis.

The therapeutic applications of RNAi are extremely broad, sinceinterfering RNA constructs can be synthesized with any nucleotidesequence directed against a target protein. To date, siRNA constructshave shown the ability to specifically down-regulate target proteins inboth in vitro and in vivo models. In addition, siRNA constructs arecurrently being evaluated in clinical studies.

However, two problems currently faced by interfering RNA constructs are,first, their susceptibility to nuclease digestion in plasma and, second,their limited ability to gain access to the intracellular compartmentwhere they can bind RISC when administered systemically as freeinterfering RNA molecules. These double-stranded constructs can bestabilized by the incorporation of chemically modified nucleotidelinkers within the molecule, e.g., phosphothioate groups. However, suchchemically modified linkers provide only limited protection fromnuclease digestion and may decrease the activity of the construct.Intracellular delivery of interfering RNA can be facilitated by the useof carrier systems such as polymers, cationic liposomes, or by thecovalent attachment of a cholesterol moiety to the molecule. However,improved delivery systems are required to increase the potency ofinterfering RNA molecules such as siRNA and miRNA and to reduce oreliminate the requirement for chemically modified nucleotide linkers.

In addition, problems remain with the limited ability of therapeuticnucleic acids such as interfering RNA to cross cellular membranes (see,Vlassov et al., Biochim. Biophys. Acta, 1197:95-1082 (1994)) and in theproblems associated with systemic toxicity, such as complement-mediatedanaphylaxis, altered coagulatory properties, and cytopenia (Galbraith etal., Antisense NucL Acid Drug Des., 4:201-206 (1994)).

To attempt to improve efficacy, investigators have also employedlipid-based carrier systems to deliver chemically modified or unmodifiedtherapeutic nucleic acids. Zelphati et al. (J. Contr. Rel., 41:99-119(1996)) describes the use of anionic (conventional) liposomes, pHsensitive liposomes, immunoliposomes, fusogenic liposomes, and cationiclipid/antisense aggregates. Similarly, siRNA has been administeredsystemically in cationic liposomes, and these nucleic acid-lipidparticles have been reported to provide improved down-regulation oftarget proteins in mammals including non-human primates (Zimmermann etal., Nature, 441: 111-114 (2006)).

In spite of this progress, there remains a need in the art for improvedlipid-therapeutic nucleic acid compositions that are suitable forgeneral therapeutic use. Preferably, these compositions wouldencapsulate nucleic acids with high efficiency, have high drug:lipidratios, protect the encapsulated nucleic acid from degradation andclearance in serum, be suitable for systemic delivery, and provideintracellular delivery of the encapsulated nucleic acid. In addition,these nucleic acid-lipid particles should be well-tolerated and providean adequate therapeutic index, such that patient treatment at aneffective dose of the nucleic acid is not associated with significanttoxicity and/or risk to the patient.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel trialkyl cationic (amino) lipidsand lipid particles comprising these lipids, which are advantageous forthe in vivo delivery of nucleic acids, as well as nucleic acid-lipidparticle compositions suitable for in vivo therapeutic use. The presentinvention also provides methods of making these compositions, as well asmethods of introducing nucleic acids into cells using thesecompositions, e.g., for the treatment of various disease conditions. Thepresent invention also includes all novel compounds and intermediatesdisclosed herein.

As described in Example 2 herein, trialkyl cationic lipids of thepresent invention are more potent, in a murine ApoB siRNA assay, thanotherwise identical lipids having longer alkyl chains.

In one aspect, the present invention provides a cationic lipid having astructural Formula (I):

X-A-Y—Z;  (I)

or salts, e.g., pharmaceutically acceptable salts, thereof, wherein:X is alkylamino;A is C₁ to C₆ optionally substituted alkyl, wherein said C₁ to C₆optionally substituted alkyl can be saturated or unsaturated, andwherein A may or may not be present;Y is selected from the group consisting of ketal, ester, optionallysubstituted carbamate, ether, and optionally substituted amide; andZ is a hydrophobic moiety consisting of three alkyl chains wherein eachof the alkyl chains has a length of from C₈ to C₁₁, wherein each of thethree alkyl chains can be saturated or unsaturated, and wherein each ofthe three alkyl chains is optionally substituted.

With respect to the lipids of Formula (I), representative examples ofalkylamino groups include dimethylamino, diethylamino, andethylmethylamino.

Again with respect to lipids of Formula (I), a representative example ofan optional substituent present on the carbamate and/or amide groups isa saturated or unsaturated alkyl group (e.g., C₁-C₆ alkyl).

Again with respect to lipids of Formula (I), a representative example ofan optional substituent that can be present on one or more of the threealkyl chains of hydrophobic moiety Z is a hydroxyl group.

Again with respect to lipids of Formula (I), it will be understood thatwhere an alkyl chain of the hydrophobic moiety Z contains one or moredouble bonds or triple bonds, then that alkyl chain is referred to asunsaturated.

Again with respect to lipids of Formula (I), it will be understood, forthe avoidance of doubt, that one or more alkyl chain of the hydrophobicmoiety Z can include a cycloalkyl group (e.g., a cyclopropyl).

Again with respect to lipids of Formula (I), it will be understood thatthe term “ester” includes esters having the structure —C(═O)O— or—OC(═O)—. The term “amide” includes amides having the structure—C(═O)NR— or —NR(═O)C—. The term “carbamate” includes carbamates havingthe structure —OC(═O)NR— or —NRC(═O)O—.

Lipids of Formula (I) are useful, for example, for making the lipidparticles of the invention which are useful, for example, for deliveringtherapeutic agents (e.g., biologically active nucleic acid molecules,such as siRNAs) to a mammal (e.g., human being) in need thereof.

In some embodiments of the lipids of Formula (I), Z has the formula:

wherein, R₁, R₂, and R₃ are each independently selected from the groupconsisting of C₈ to C₁₁ alkyl, wherein each of R₁, R₂, and R₃ canindependently be saturated or unsaturated, and wherein each of R₁, R₂,and R₃ is optionally substituted.

In a further aspect, the present invention provides a lipid particlecomprising one or more of the above cationic lipids of Formula I orsalts, e.g., pharmaceutically acceptable salts, thereof. In certainembodiments, the lipid particle further comprises one or morenon-cationic lipids such as neutral lipids. In certain otherembodiments, the lipid particle further comprises one or more conjugatedlipids capable of reducing or inhibiting particle aggregation. Inadditional embodiments, the lipid particle further comprises one or moreactive agents or therapeutic agents.

In certain embodiments, the non-cationic lipid component of the lipidparticle may comprise a phospholipid, cholesterol (or cholesterolderivative), or a mixture thereof. In one particular embodiment, thephospholipid comprises dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine (DSPC), or a mixture thereof. In someembodiments, the conjugated lipid component of the lipid particlecomprises a polyethyleneglycol (PEG)-lipid conjugate. In certaininstances, the PEG-lipid conjugate comprises a PEG-diacylglycerol(PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or amixture thereof. In other embodiments, the lipid conjugate comprises apolyoxazoline (POZ)-lipid conjugate such as a POZ-DAA conjugate.

In some embodiments, the active agent or therapeutic agent comprises anucleic acid. In certain instances, the nucleic acid comprises aninterfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA,Dicer-substrate dsRNA, shRNA, or mixtures thereof. In certain otherinstances, the nucleic acid comprises single-stranded or double-strandedDNA, RNA, or a DNA/RNA hybrid such as, e.g., an antisenseoligonucleotide, a ribozyme, a plasmid, an immunostimulatoryoligonucleotide, or mixtures thereof.

In other embodiments, the active agent or therapeutic agent is fullyencapsulated within the lipid portion of the lipid particle such thatthe active agent or therapeutic agent in the lipid particle is resistantin aqueous solution to enzymatic degradation, e.g., by a nuclease orprotease. In further embodiments, the lipid particle is substantiallynon-toxic to mammals such as humans.

In certain embodiments, the present invention provides lipid particles(e.g., LNP) comprising: (a) one or more nucleic acids such asinterfering RNA molecules; (b) one or more cationic lipids of Formula Ior salts, e.g., pharmaceutically acceptable salts, thereof; (c) one ormore non-cationic lipids; and (d) one or more conjugated lipids thatinhibit aggregation of particles.

In some embodiments, the present invention provides lipid particles(e.g., LNP) comprising: (a) one or more nucleic acids; (b) one or morecationic lipids of Formula I or salts, e.g., pharmaceutically acceptablesalts, thereof comprising from about 50 mol % to about 85 mol % of thetotal lipid present in the particle; (c) one or more non-cationic lipidscomprising from about 13 mol % to about 49.5 mol % of the total lipidpresent in the particle; and (d) one or more conjugated lipids thatinhibit aggregation of particles comprising from about 0.5 mol % toabout 2 mol % of the total lipid present in the particle.

In one aspect of this embodiment, the lipid particle (e.g., LNP)comprises: (a) a nucleic acid; (b) a cationic lipid of Formula I or asalt, e.g., a pharmaceutically acceptable salt, thereof comprising fromabout 52 mol % to about 62 mol % of the total lipid present in theparticle; (c) a mixture of a phospholipid and cholesterol or aderivative thereof comprising from about 36 mol % to about 47 mol % ofthe total lipid present in the particle; and (d) a PEG-lipid conjugatecomprising from about 1 mol % to about 2 mol % of the total lipidpresent in the particle. This embodiment of nucleic acid-lipid particleis generally referred to herein as the “1:57” formulation. In oneparticular embodiment, the 1:57 formulation is a four-component systemcomprising about 1.4 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA),about 57.1 mol % cationic lipid of Formula I or a salt thereof, about7.1 mol % DPPC (or DSPC), and about 34.3 mol % cholesterol (orderivative thereof).

In another aspect of this embodiment, the lipid particle (e.g., LNP)comprises: (a) a nucleic acid; (b) a cationic lipid of Formula I or asalt, e.g., a pharmaceutically acceptable salt, thereof comprising fromabout 56.5 mol % to about 66.5 mol % of the total lipid present in theparticle; (c) cholesterol or a derivative thereof comprising from about31.5 mol % to about 42.5 mol % of the total lipid present in theparticle; and (d) a PEG-lipid conjugate comprising from about 1 mol % toabout 2 mol % of the total lipid present in the particle. Thisembodiment of nucleic acid-lipid particle is generally referred toherein as the “1:62” formulation. In one particular embodiment, the 1:62formulation is a three-component system which is phospholipid-free andcomprises about 1.5 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA),about 61.5 mol % cationic lipid of Formula I or a salt thereof, andabout 36.9 mol % cholesterol (or derivative thereof).

Additional embodiments related to the 1:57 and 1:62 formulations aredescribed in PCT Publication No. WO 09/127060, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

In other embodiments, the present invention provides lipid particles(e.g., LNP) comprising: (a) one or more nucleic acids; (b) one or morecationic lipids of Formula I or II or salts, e.g., pharmaceuticallyacceptable salts, thereof comprising from about 2 mol % to about 50 mol% of the total lipid present in the particle; (c) one or morenon-cationic lipids comprising from about 5 mol % to about 90 mol % ofthe total lipid present in the particle; and (d) one or more conjugatedlipids that inhibit aggregation of particles comprising from about 0.5mol % to about 20 mol % of the total lipid present in the particle.

In one aspect of this embodiment, the lipid particle (e.g., LNP)comprises: (a) a nucleic acid; (b) a cationic lipid of Formula I or asalt, e.g., a pharmaceutically acceptable salt, thereof comprising fromabout 30 mol % to about 50 mol % of the total lipid present in theparticle; (c) a mixture of a phospholipid and cholesterol or aderivative thereof comprising from about 47 mol % to about 69 mol % ofthe total lipid present in the particle; and (d) a PEG-lipid conjugatecomprising from about 1 mol % to about 3 mol % of the total lipidpresent in the particle. This embodiment of lipid particle is generallyreferred to herein as the “2:40” formulation. In one particularembodiment, the 2:40 formulation is a four-component system whichcomprises about 2 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA), about40 mol % cationic lipid of Formula I or a salt thereof, about 10 mol %DPPC (or DSPC), and about 48 mol % cholesterol (or derivative thereof).

In further embodiments, the present invention provides nucleicacid-lipid particles (e.g., LNP) comprising: (a) one or more nucleicacids; (b) one or more cationic lipids of Formula I or salts, e.g.,pharmaceutically acceptable salts, thereof comprising from about 50 mol% to about 65 mol % of the total lipid present in the particle; (c) oneor more non-cationic lipids comprising from about 25 mol % to about 45mol % of the total lipid present in the particle; and (d) one or moreconjugated lipids that inhibit aggregation of particles comprising fromabout 5 mol % to about 10 mol % of the total lipid present in theparticle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) a nucleic acid; (b) a cationic lipid of Formula I or asalt, e.g., a pharmaceutically acceptable salt, thereof comprising fromabout 50 mol % to about 60 mol % of the total lipid present in theparticle; (c) a mixture of a phospholipid and cholesterol or aderivative thereof comprising from about 35 mol % to about 45 mol % ofthe total lipid present in the particle; and (d) a PEG-lipid conjugatecomprising from about 5 mol % to about 10 mol % of the total lipidpresent in the particle. This embodiment of nucleic acid-lipid particleis generally referred to herein as the “7:54” formulation. In certaininstances, the non-cationic lipid mixture in the 7:54 formulationcomprises: (i) a phospholipid of from about 5 mol % to about 10 mol % ofthe total lipid present in the particle; and (ii) cholesterol or aderivative thereof of from about 25 mol % to about 35 mol % of the totallipid present in the particle. In one particular embodiment, the 7:54formulation is a four-component system which comprises about 7 mol %PEG-lipid conjugate (e.g., PEG750-C-DMA), about 54 mol % cationic lipidof Formula I or a salt thereof, about 7 mol % DPPC (or DSPC), and about32 mol % cholesterol (or derivative thereof).

In another aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) a nucleic acid; (b) a cationic lipid of Formula I or asalt, e.g., a pharmaceutically acceptable salt, thereof comprising fromabout 55 mol % to about 65 mol % of the total lipid present in theparticle; (c) cholesterol or a derivative thereof comprising from about30 mol % to about 40 mol % of the total lipid present in the particle;and (d) a PEG-lipid conjugate comprising from about 5 mol % to about 10mol % of the total lipid present in the particle. This embodiment ofnucleic acid-lipid particle is generally referred to herein as the“7:58” formulation. In one particular embodiment, the 7:58 formulationis a three-component system which is phospholipid-free and comprisesabout 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about 58 mol %cationic lipid of Formula I or a salt thereof, and about 35 mol %cholesterol (or derivative thereof).

Additional embodiments related to the 7:54 and 7:58 formulations aredescribed in U.S. Published Patent Application No. US2011/0076335, filedJun. 30, 2010, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

The present invention also provides pharmaceutical compositionscomprising a lipid particle such as a nucleic acid-lipid particle (e.g.,LNP) and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides methods forintroducing one or more therapeutic agents such as nucleic acids into acell, the method comprising contacting the cell with a lipid particledescribed herein (e.g., LNP). In one embodiment, the cell is in a mammaland the mammal is a human.

In yet another aspect, the present invention provides methods for the invivo delivery of one or more therapeutic agents such as nucleic acids,the method comprising administering to a mammal a lipid particledescribed herein (e.g., LNP). In certain embodiments, the lipidparticles (e.g., LNP) are administered by one of the following routes ofadministration: oral, intranasal, intravenous, intraperitoneal,intramuscular, intra-articular, intralesional, intratracheal,subcutaneous, and intradermal. In particular embodiments, the lipidparticles (e.g., LNP) are administered systemically, e.g., via enteralor parenteral routes of administration. In preferred embodiments, themammal is a human.

In a further aspect, the present invention provides methods for treatinga disease or disorder in a mammal in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of alipid particle (e.g., LNP) comprising one or more therapeutic agentssuch as nucleic acids. Non-limiting examples of diseases or disordersinclude a viral infection, a liver disease or disorder, and cancer.Preferably, the mammal is a human.

In certain embodiments, the present invention provides methods fortreating a liver disease or disorder by administering a nucleic acidsuch as an interfering RNA (e.g., siRNA) in nucleic acid-lipid particles(e.g., LNP), alone or in combination with a lipid-lowering agent.Examples of lipid diseases and disorders include, but are not limitedto, dyslipidemia (e.g., hyperlipidemias such as elevated triglyceridelevels (hypertriglyceridemia) and/or elevated cholesterol levels(hypercholesterolemia)), atherosclerosis, coronary heart disease,coronary artery disease, atherosclerotic cardiovascular disease (CVD),fatty liver disease (hepatic steatosis), abnormal lipid metabolism,abnormal cholesterol metabolism, diabetes (including Type 2 diabetes),obesity, cardiovascular disease, and other disorders relating toabnormal metabolism. Non-limiting examples of lipid-lowering agentsinclude statins, fibrates, ezetimibe, thiazolidinediones, niacin,beta-blockers, nitroglycerin, calcium antagonists, and fish oil.

In one particular embodiment, the present invention provides a methodfor lowering or reducing cholesterol levels in a mammal (e.g., human) inneed thereof (e.g., a mammal with elevated blood cholesterol levels),the method comprising administering to the mammal a therapeuticallyeffective amount of a nucleic acid-lipid particle (e.g., a LNPformulation) described herein comprising one or more interfering RNAs(e.g., siRNAs) that target one or more genes associated with metabolicdiseases and disorders. In another particular embodiment, the presentinvention provides a method for lowering or reducing triglyceride levelsin a mammal (e.g., human) in need thereof (e.g., a mammal with elevatedblood triglyceride levels), the method comprising administering to themammal a therapeutically effective amount of a nucleic acid-lipidparticle (e.g., a LNP formulation) described herein comprising one ormore interfering RNAs (e.g., siRNAs) that target one or more genesassociated with metabolic diseases and disorders. These methods can becarried out in vitro using standard tissue culture techniques or in vivoby administering the interfering RNA (e.g., siRNA) using any means knownin the art. In preferred embodiments, the interfering RNA (e.g., siRNA)is delivered to a liver cell (e.g., hepatocyte) in a mammal such as ahuman.

Additional embodiments related to treating a liver disease or disorderusing a lipid particle are described in, e.g., PCT Application No.PCT/CA2010/000120, filed Jan. 26, 2010, and U.S. Patent ApplicationPublication No. 2006/0134189, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

In other embodiments, the present invention provides methods fortreating a cell proliferative disorder such as cancer by administering anucleic acid such as an interfering RNA (e.g., siRNA) in nucleicacid-lipid particles (e.g., LNP), alone or in combination with achemotherapy drug. The methods can be carried out in vitro usingstandard tissue culture techniques or in vivo by administering theinterfering RNA (e.g., siRNA) using any means known in the art. Inpreferred embodiments, the interfering RNA (e.g., siRNA) is delivered toa cancer cell in a mammal such as a human, alone or in combination witha chemotherapy drug. The nucleic acid-lipid particles and/orchemotherapy drugs may also be co-administered with conventionalhormonal, immunotherapeutic, and/or radiotherapeutic agents.

Additional embodiments related to treating a cell proliferative disorderusing a lipid particle are described in, e.g., PCT Publication No. WO09/082817, U.S. Patent Application Publication No. 2009/0149403, and PCTPublication No. WO 09/129319, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

In further embodiments, the present invention provides methods forpreventing or treating a viral infection such as an arenavirus (e.g.,Lassa virus) or filovirus (e.g., Ebola virus, Marburg virus, etc.)infection which causes hemorrhagic fever or a hepatitis (e.g., HepatitisC virus) infection which causes acute or chronic hepatitis byadministering a nucleic acid such as an interfering RNA (e.g., siRNA) innucleic acid-lipid particles (e.g., LNP), alone or in combination withthe administration of conventional agents used to treat or amelioratethe viral condition or any of the symptoms associated therewith. Themethods can be carried out in vitro using standard tissue culturetechniques or in vivo by administering the interfering RNA using anymeans known in the art. In certain embodiments, the interfering RNA(e.g., siRNA) is delivered to cells, tissues, or organs of a mammal suchas a human that are infected and/or susceptible of being infected withthe hemorrhagic fever virus, such as, e.g., cells of thereticuloendothelial system (e.g., monocytes, macrophages, etc.). Incertain other embodiments, the interfering RNA (e.g., siRNA) isdelivered to cells, tissues, or organs of a mammal such as a human thatare infected and/or susceptible of being infected with the hepatitisvirus, such as, e.g., cells of the liver (e.g., hepatocytes).

Additional embodiments related to preventing or treating a viralinfection using a lipid particle are described in, e.g., U.S. PatentApplication Publication No. 2007/0218122, U.S. Patent ApplicationPublication No. 2007/0135370, and PCT Application No. PCT/CA2010/000444,entitled “Compositions and Methods for Silencing Hepatitis C VirusExpression,” filed Mar. 19, 2010, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

The lipid particles of the invention (e.g., LNP), comprising one or morecationic lipids of Formula I or salts, e.g., pharmaceutically acceptablesalts, thereof, are particularly advantageous and suitable for use inthe administration of nucleic acids such as interfering RNA to a subject(e.g., a mammal such as a human) because they are stable in circulation,of a size required for pharmacodynamic behavior resulting in access toextravascular sites, and are capable of reaching target cellpopulations.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and FIGURES.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based, in part, upon the discovery of novelcationic (amino) lipids that provide advantages when used in lipidparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular, thepresent invention provides nucleic acid-lipid particle compositionscomprising one or more of the novel cationic lipids described hereinthat provide increased activity of the nucleic acid (e.g., interferingRNA) and improved tolerability of the compositions in vivo, resulting ina significant increase in the therapeutic index as compared to nucleicacid-lipid particle compositions previously described.

In particular embodiments, the present invention provides novel cationiclipids that enable the formulation of improved compositions for the invitro and in vivo delivery of interfering RNA such as siRNA. It is shownherein that these improved lipid particle compositions are effective indown-regulating (e.g., silencing) the protein levels and/or mRNA levelsof target genes. Furthermore, it is shown herein that the activity ofthese improved lipid particle compositions is dependent on the presenceof the novel cationic lipids of the invention.

The lipid particles and compositions of the present invention may beused for a variety of purposes, including the delivery of encapsulatedor associated (e.g., complexed) therapeutic agents such as nucleic acidsto cells, both in vitro and in vivo. Accordingly, the present inventionfurther provides methods of treating diseases or disorders in a subjectin need thereof by contacting the subject with a lipid particle thatencapsulates or is associated with a suitable therapeutic agent, whereinthe lipid particle comprises one or more of the novel cationic lipidsdescribed herein.

As described herein, the lipid particles of the present invention areparticularly useful for the delivery of nucleic acids, including, e.g.,interfering RNA molecules such as siRNA. Therefore, the lipid particlesand compositions of the present invention may be used to decrease theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid particle comprising one or more novelcationic lipids described herein, wherein the lipid particleencapsulates or is associated with a nucleic acid that reduces targetgene expression (e.g., an siRNA). Alternatively, the lipid particles andcompositions of the present invention may be used to increase theexpression of a desired protein both in vitro and in vivo by contactingcells with a lipid particle comprising one or more novel cationic lipidsdescribed herein, wherein the lipid particle encapsulates or isassociated with a nucleic acid that enhances expression of the desiredprotein (e.g., a plasmid encoding the desired protein).

Various exemplary embodiments of the cationic lipids of the presentinvention, lipid particles and compositions comprising the same, andtheir use to deliver active or therapeutic agents such as nucleic acidsto modulate gene and protein expression, are described in further detailbelow.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “about” when used in connection with the amount of a componentin a lipid particle or formulation of the present invention encompassesvalues that are plus or minus 5% of the stated amount of the component(e.g., about 10% encompasses values of from 9.5% to 10.5%). The term“about” therefore also encompasses values that are plus or minus 1%, 2%,3%, or 4% of the stated amount of the component.

The term “interfering RNA” or “RNAi” or “interfering RNA sequence” asused herein includes single-stranded RNA (e.g., mature miRNA, ssRNAioligonucleotides, ssDNAi oligonucleotides) or double-stranded RNA (i.e.,duplex RNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, orpre-miRNA) that is capable of reducing or inhibiting the expression of atarget gene or sequence (e.g., by mediating the degradation orinhibiting the translation of mRNAs which are complementary to theinterfering RNA sequence) when the interfering RNA is in the same cellas the target gene or sequence. Interfering RNA thus refers to thesingle-stranded RNA that is complementary to a target mRNA sequence orto the double-stranded RNA formed by two complementary strands or by asingle, self-complementary strand. Interfering RNA may have substantialor complete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). The sequence of theinterfering RNA can correspond to the full-length target gene, or asubsequence thereof. Preferably, the interfering RNA molecules arechemically synthesized.

Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g.,interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides inlength, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, and is preferably about 20-24, 21-22, or 21-23 (duplex)nucleotides in length (e.g., each complementary sequence of thedouble-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25nucleotides in length, preferably about 20-24, 21-22, or 21-23nucleotides in length, and the double-stranded siRNA is about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferablyabout 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes maycomprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 toabout 3 nucleotides and 5′ phosphate termini. Examples of siRNA include,without limitation, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingself-complementary sense and antisense regions; and a circularsingle-stranded polynucleotide molecule with two or more loop structuresand a stem having self-complementary sense and antisense regions, wherethe circular polynucleotide can be processed in vivo or in vitro togenerate an active double-stranded siRNA molecule.

Preferably, siRNA are chemically synthesized. siRNA can also begenerated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25nucleotides in length) with the E. coli RNase III or Dicer. Theseenzymes process the dsRNA into biologically active siRNA (see, e.g.,Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegariet al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al.,Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res.,31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); andRobertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA areat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotidesin length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotidesin length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that automatically foldinto duplexes with hairpin loops).

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an interfering RNA (e.g., siRNA) sequence that does nothave 100% complementarity to its target sequence. An interfering RNA mayhave at least one, two, three, four, five, six, or more mismatchregions. The mismatch regions may be contiguous or may be separated by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatchmotifs or regions may comprise a single nucleotide or may comprise two,three, four, five, or more nucleotides.

The phrase “inhibiting expression of a target gene” refers to theability of an interfering RNA (e.g., siRNA), or another therapeuticagent, to silence, reduce, or inhibit the expression of a target gene.To examine the extent of gene silencing, a test sample (e.g., a sampleof cells in culture expressing the target gene) or a test mammal (e.g.,a mammal such as a human or an animal model such as a rodent (e.g.,mouse) or a non-human primate (e.g., monkey) model) is contacted with aninterfering RNA (e.g., siRNA) that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample or test animal is compared to expression of the target gene in acontrol sample (e.g., a sample of cells in culture expressing the targetgene) or a control mammal (e.g., a mammal such as a human or an animalmodel such as a rodent (e.g., mouse) or non-human primate (e.g., monkey)model) that is not contacted with or administered the interfering RNA(e.g., siRNA). The expression of the target gene in a control sample ora control mammal may be assigned a value of 100%. In particularembodiments, silencing, inhibition, or reduction of expression of atarget gene is achieved when the level of target gene expression in thetest sample or the test mammal relative to the level of target geneexpression in the control sample or the control mammal is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. In other words, the interfering RNA (e.g.,siRNA) silences, reduces, or inhibits the expression of a target gene byat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or atest mammal relative to the level of target gene expression in a controlsample or a control mammal not contacted with or administered theinterfering RNA (e.g., siRNA). Suitable assays for determining the levelof target gene expression include, without limitation, examination ofprotein or mRNA levels using techniques known to those of skill in theart, such as, e.g., dot blots, Northern blots, in situ hybridization,ELISA, immunoprecipitation, enzyme function, as well as phenotypicassays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as an interfering RNA is an amountsufficient to produce the desired effect, e.g., an inhibition ofexpression of a target sequence in comparison to the normal expressionlevel detected in the absence of an interfering RNA. Inhibition ofexpression of a target gene or target sequence is achieved when thevalue obtained with an interfering RNA relative to the control is about95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%,25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expressionof a target gene or target sequence include, e.g., examination ofprotein or RNA levels using techniques known to those of skill in theart such as dot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, as well as phenotypic assays knownto those of skill in the art.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by an interfering RNA is intended to mean a detectable decreaseof an immune response to a given interfering RNA (e.g., a modifiedinterfering RNA) or other therapeutic agent. The amount of decrease ofan immune response by a modified interfering RNA may be determinedrelative to the level of an immune response in the presence of anunmodified interfering RNA. A detectable decrease can be about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, or more lower than the immune response detected inthe presence of the unmodified interfering RNA. A decrease in the immuneresponse to interfering RNA is typically measured by a decrease incytokine production (e.g., IFNγ, IFNα, TNFα, IL-6, or IL-12) by aresponder cell in vitro or a decrease in cytokine production in the seraof a mammalian subject after administration of the interfering RNA.

As used herein, the term “responder cell” refers to a cell, preferably amammalian cell, that produces a detectable immune response whencontacted with an immunostimulatory interfering RNA such as anunmodified siRNA. Exemplary responder cells include, e.g., dendriticcells, macrophages, peripheral blood mononuclear cells (PBMCs),splenocytes, and the like. Detectable immune responses include, e.g.,production of cytokines or growth factors such as TNF-α, IFN-α, IFN-γ,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, andcombinations thereof. Detectable immune responses also include, e.g.,induction of interferon-induced protein with tetratricopeptide repeats 1(IFIT1) mRNA.

“Substantial identity” refers to a sequence that hybridizes to areference sequence under stringent conditions, or to a sequence that hasa specified percent identity over a specified region of a referencesequence.

The phrase “stringent hybridization conditions” refers to conditionsunder which a nucleic acid will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m), is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m) 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization.

Exemplary stringent hybridization conditions can be as follows: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec.-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y. (1990).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same (i.e., at least about 60%, preferably atleast about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. This definition, when the context indicates, alsorefers analogously to the complement of a sequence. Preferably, thesubstantial identity exists over a region that is at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of a number of contiguous positions selected from the groupconsisting of from about 5 to about 60, usually about 10 to about 45,more usually about 15 to about 30, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch, J Mol. Biol.,48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology, Ausubelet al., eds. (1995 supplement)).

Non-limiting examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.,215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). Anotherexample is a global alignment algorithm for determining percent sequenceidentiy such as the Needleman-Wunsch algorithm for aligning protein ornucleotide (e.g., RNA) sequences.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA and RNA. DNA may be in the formof, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCRproduct, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expressioncassettes, chimeric sequences, chromosomal DNA, or derivatives andcombinations of these groups. RNA may be in the form of smallinterfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA(shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA,tRNA, rRNA, tRNA, viral RNA (vRNA), multivalent RNA (MV RNA), andcombinations thereof. Nucleic acids include nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,and which have similar binding properties as the reference nucleic acid.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, SNPs, and complementary sequences aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

The term “lipid particle” includes a lipid formulation that can be usedto deliver an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA), to a target site of interest (e.g., cell,tissue, organ, and the like). In preferred embodiments, the lipidparticle of the invention is a lipid nanoparticle, which is typicallyformed from a cationic lipid, a non-cationic lipid, and optionally aconjugated lipid that prevents aggregation of the particle. In otherpreferred embodiments, that may be referred to as “nucleic acid-lipidparticles”, the active agent or therapeutic agent, such as a nucleicacid, may be encapsulated in the lipid portion of the particle, therebyprotecting it from enzymatic degradation.

As used herein, the term “LNP” refers to a lipid nanoparticle. An LNPrepresents a particle made from lipids (e.g., a cationic lipid, anon-cationic lipid, and optionally a conjugated lipid that preventsaggregation of the particle), wherein the nucleic acid (e.g., aninterfering RNA) is fully encapsulated within the lipid. In certaininstances, LNP are extremely useful for systemic applications, as theycan exhibit extended circulation lifetimes following intravenous (i.v.)injection, they can accumulate at distal sites (e.g., sites physicallyseparated from the administration site), and they can mediate silencingof target gene expression at these distal sites. The nucleic acid may becomplexed with a condensing agent and encapsulated within an LNP as setforth in PCT Publication No. WO 00/03683, the disclosure of which isherein incorporated by reference in its entirety for all purposes.

The lipid particles of the invention (e.g., LNP) typically have a meandiameter of from about 30 nm to about 150 nm, from about 40 nm to about150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm,from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In addition, nucleic acids, whenpresent in the lipid particles of the present invention, are resistantin aqueous solution to degradation with a nuclease. Lipid nanoparticlesand their method of preparation are disclosed in, e.g., U.S. PatentApplication Publication Nos. 2004/0142025 and 2007/0042031, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

As used herein, “lipid encapsulated” can refer to a lipid particle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA), with full encapsulation, partialencapsulation, or both. In a preferred embodiment, the nucleic acid isfully encapsulated in the lipid particle (e.g., to form an LNP).

The term “lipid conjugate” refers to a conjugated lipid that inhibitsaggregation of lipid particles. Such lipid conjugates include, but arenot limited to, PEG-lipid conjugates such as, e.g., PEG coupled todialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled todiacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol,PEG coupled to phosphatidylethanolamines, and PEG conjugated toceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids,polyoxazoline (POZ)-lipid conjugates, polyamide oligomers (e.g.,ATTA-lipid conjugates), and mixtures thereof. Additional examples ofPOZ-lipid conjugates are described in PCT Publication No. WO2010/006282. PEG or POZ can be conjugated directly to the lipid or maybe linked to the lipid via a linker moiety. Any linker moiety suitablefor coupling the PEG or the POZ to a lipid can be used including, e.g.,non-ester containing linker moieties and ester-containing linkermoieties. In certain preferred embodiments, non-ester containing linkermoieties, such as amides or carbamates, are used. The disclosures ofeach of the above patent documents are herein incorporated by referencein their entirety for all purposes.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The term “fusogenic” refers to the ability of a lipid particle, such asa LNP, to fuse with the membranes of a cell. The membranes can be eitherthe plasma membrane or membranes surrounding organelles, e.g., endosome,nucleus, etc.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles such as LNPmeans that the particle is not significantly degraded after exposure toa serum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent suchas an interfering RNA (e.g., siRNA) within an organism. Some techniquesof administration can lead to the systemic delivery of certain agents,but not others. Systemic delivery means that a useful, preferablytherapeutic, amount of an agent is exposed to most parts of the body. Toobtain broad biodistribution generally requires a blood lifetime suchthat the agent is not rapidly degraded or cleared (such as by first passorgans (liver, lung, etc.) or by rapid, nonspecific cell binding) beforereaching a disease site distal to the site of administration. Systemicdelivery of lipid particles can be by any means known in the artincluding, for example, intravenous, subcutaneous, and intraperitoneal.In a preferred embodiment, systemic delivery of lipid particles is byintravenous delivery.

“Local delivery,” as used herein, refers to delivery of an active agentsuch as an interfering RNA (e.g., siRNA) directly to a target sitewithin an organism. For example, an agent can be locally delivered bydirect injection into a disease site such as a tumor or other targetsite such as a site of inflammation or a target organ such as the liver,heart, pancreas, kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

The term “cancer” refers to any member of a class of diseasescharacterized by the uncontrolled growth of aberrant cells. The termincludes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,liver cancer, lung cancer, colon cancer, rectal cancer, anal cancer,bile duct cancer, small intestine cancer, stomach (gastric) cancer,esophageal cancer; gallbladder cancer, pancreatic cancer, appendixcancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer,renal cancer (e.g., renal cell carcinoma), cancer of the central nervoussystem, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head andneck cancers, osteogenic sarcomas, and blood cancers. Non-limitingexamples of specific types of liver cancer include hepatocellularcarcinoma (HCC), secondary liver cancer (e.g., caused by metastasis ofsome other non-liver cancer cell type), and hepatoblastoma. As usedherein, a “tumor” comprises one or more cancerous cells.

The term “Multivalent RNA”, abbreviated as “MV RNA”, refers to apolynucleotide complex composed of at least three polynucleotides,wherein each polynucleotide is hybridized, along all or part of itslength, to at least two of the other polynucleotides of the complex andwherein one or more of the polynucleotides optionally includes atargeting region that is capable of hybridizing to a target nucleic acidsequence. Each polynucleotide can be, for example, from 10 to 60nucleotides in length. The targeting region(s) within a polynucleotidecan be capable of hybridizing to a target nucleic acid sequence that isthe same or different than the target nucleic acid sequence(s) to whichthe targeting region(s) of the other polynucleotides of the complexhybridize. A Multivalent RNA may be synthesized in vitro (e.g., bychemical synthesis) or, for example, it may be processed from aprecursor within a living cell. For example, a precursor can be a linearpolynucleotide that includes each of the polynucleotides of theMultivalent RNA, which is introduced into a living cell and is cleavedtherein to form a Multivalent RNA. The term “Multivalent RNA” includessuch a precursor that is intended to be cleaved inside a living cell.The term “Multivalent RNA” also encompasses, by way of example, thetripartite polynucleotide complexes described, specifically orgenerically, in the published international patent application havinginternational application number PCT/US2010/036962.

III. Novel Cationic Lipids

The present invention provides, inter alia, novel cationic (amino)lipids that can advantageously be used in the lipid particles describedherein for the in vitro and/or in vivo delivery of therapeutic agentssuch as nucleic acids to cells. The novel cationic lipids of theinvention have the structures set forth in Formula I herein, and includethe (R) and/or (S) enantiomers thereof.

In some embodiments, a lipid of the present invention comprises aracemic mixture. In other embodiments, a lipid of the present inventioncomprises a mixture of one or more diastereomers. In certainembodiments, a lipid of the present invention is enriched in oneenantiomer, such that the lipid comprises at least about 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% enantiomeric excess. In certain otherembodiments, a lipid of the present invention is enriched in onediastereomer, such that the lipid comprises at least about 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% diastereomeric excess. In certainadditional embodiments, a lipid of the present invention is chirallypure (e.g., comprises a single optical isomer). In further embodiments,a lipid of the present invention is enriched in one optical isomer(e.g., an optically active isomer), such that the lipid comprises atleast about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isomericexcess. The present invention provides the synthesis of the cationiclipids of Formula I as a racemic mixture or in optically pure form.

The terms “cationic lipid” and “amino lipid” are used interchangeablyherein to include those lipids and salts, e.g., pharmaceuticallyacceptable salts, thereof having one, two, three, or more fatty acid orfatty alkyl chains and a pH-titratable amino head group (e.g., analkylamino or dialkylamino head group). The cationic lipid is typicallyprotonated (i.e., positively charged) at a pH below the pK_(a) of thecationic lipid and is substantially neutral at a pH above the pK_(a).The cationic lipids of the invention may also be termed titratablecationic lipids.

The term “salts” includes any anionic and cationic complex, such as thecomplex formed between a cationic lipid disclosed herein and one or moreanions. Non-limiting examples of anions include inorganic and organicanions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate(e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate,dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite,nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate,hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate,lactate, acrylate, polyacrylate, fumarate, maleate, itaconate,glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate,polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite,bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate,arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate,hydroxide, peroxide, permanganate, and mixtures thereof. In particularembodiments, the salts of the cationic lipids disclosed herein arecrystalline salts. In particular embodiments, “salts” are“pharmaceutically acceptable salts.”

The term “pharmaceutically acceptable salts” refers to pharmaceuticallyacceptable salts of a compound, which salts are derived from a varietyof organic and inorganic counter ions well known in the art.Pharmaceutically acceptable salts include both the metallic (inorganic)salts and organic salts, including but not limited to those listed inRemington's Pharmaceutical Sciences, 17th Edition, pg. 1418 (1985).Pharmaceutically acceptable salts include, by way of example only, saltsof inorganic acids such as hydrochloride, sulfate, phosphate,diphosphate, hydrobromide, and nitrate or salts of an organic acid suchas malate, maleate, fumarate, tartrate, succinate, citrate, acetate,lactate, methanesulfonate, p-toluenesulfonate or palmoate, salicylateand stearate. Similarly pharmaceutically acceptable cations include, butare not limited to, sodium, potassium, calcium, aluminum, lithium andammonium (especially ammonium salts with secondary amines). Particularsalts of this invention for the reasons cited above include potassium,sodium, calcium and ammonium salts.

The term “alkyl” includes a straight chain or branched, noncyclic orcyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbonatoms. Representative saturated straight chain alkyls include, but arenot limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, andthe like, while saturated branched alkyls include, without limitation,isopropyl, sec-butyl, isobutyl, text-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include, but are not limited to,the _(C3-8) cycloalkyls described herein, while unsaturated cyclicalkyls include, without limitation, the _(C3-8) cycloalkenyls describedherein.

The term “heteroalkyl,” includes a straight chain or branched, noncyclicor cyclic, saturated aliphatic hydrocarbon as defined above having fromabout 1 to about 5 heteroatoms (i.e., 1, 2, 3, 4, or 5 heteroatoms) suchas, for example, O, N, Si, and/or S, wherein the nitrogen and sulfuratoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroalkyl group can be attached to theremainder of the molecule through a carbon atom or a heteroatom.

The term “cyclic alkyl” includes any of the substituted or unsubstitutedcycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenylgroups described below.

The term “cycloalkyl” includes a substituted or unsubstituted cyclicalkyl group having from about 3 to about 8 carbon atoms (i.e., 3, 4, 5,6, 7, or 8 carbon atoms) as ring vertices. Preferred cycloalkyl groupsinclude those having from about 3 to about 6 carbon atoms as ringvertices. Examples of C₃₋₈ cycloalkyl groups include, but are notlimited to, cyclopropyl, methyl-cyclopropyl, dimethyl-cyclopropyl,cyclobutyl, methyl-cyclobutyl, cyclopentyl, methyl-cyclopentyl,cyclohexyl, methyl-cyclohexyl, dimethyl-cyclohexyl, cycloheptyl, andcyclooctyl, as well as other substituted C₃₋₈ cycloalkyl groups.

The term “heterocycloalkyl” includes a substituted or unsubstitutedcyclic alkyl group as defined above having from about 1 to about 3heteroatoms as ring members selected from the group consisting of O, N,Si and S, wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheterocycloalkyl group can be attached to the remainder of the moleculethrough a carbon atom or a heteroatom.

The term “cycloalkenyl” includes a substituted or unsubstituted cyclicalkenyl group having from about 3 to about 8 carbon atoms (i.e., 3, 4,5, 6, 7, or 8 carbon atoms) as ring vertices. Preferred cycloalkenylgroups are those having from about 3 to about 6 carbon atoms as ringvertices. Examples of C₃₋₈ cycloalkenyl groups include, but are notlimited to, cyclopropenyl, methyl-cyclopropenyl, dimethyl-cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, andcyclooctenyl, as well as other substituted C₃₋₈ cycloalkenyl groups.

The term “heterocycloalkenyl” includes a substituted or unsubstitutedcyclic alkenyl group as defined above having from about 1 to about 3heteroatoms as ring members selected from the group consisting of O, N,Si and S, wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheterocycloalkenyl group can be attached to the remainder of themolecule through a carbon atom or a heteroatom.

The term “alkoxy” includes a group of the formula alkyl-O—, wherein“alkyl” has the previously given definition. Non-limiting examples ofalkoxy groups include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,iso-butoxy, sec-butoxy and tert-butoxy.

The term “alkenyl” includes an alkyl, as defined above, containing atleast one double bond between adjacent carbon atoms. Alkenyls includeboth cis and trans isomers. Representative straight chain and branchedalkenyls include, but are not limited to, ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike. Representative cyclic alkenyls are described above.

The term “alkynyl” includes any alkyl or alkenyl, as defined above,which additionally contains at least one triple bond between adjacentcarbons. Representative straight chain and branched alkynyls include,without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

The term “aryl” includes a polyunsaturated, typically aromatic,hydrocarbon group which can be a single ring or multiple rings (up tothree rings) which are fused together or linked covalently, and whichoptionally carries one or more substituents, such as, for example,halogen, trifluoromethyl, amino, alkyl, alkoxy, alkylcarbonyl, cyano,carbamoyl, alkoxycarbamoyl, methylendioxy, carboxy, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, nitro,and the like. Non-limiting examples of unsubstituted aryl groups includephenyl, naphthyl, and biphenyl. Examples of substituted aryl groupsinclude, but are not limited to, phenyl, chlorophenyl,trifluoromethylphenyl, chlorofluorophenyl, and aminophenyl.

The terms “alkylthio,” “alkylsulfonyl,” “alkylsulfinyl,” and“arylsulfonyl” include groups having the formula —S(O)₂—R^(i),—S(O)—R^(i) and —S(O)₂R^(j), respectively, wherein R^(i) is an alkylgroup as previously defined and R^(j) is an aryl group as previouslydefined.

The terms “alkenyloxy” and “alkynyloxy” include groups having theformula —O—R^(i), wherein R^(i) is an alkenyl or alkynyl group,respectively.

The terms “alkenylthio” and “alkynylthio” include groups having theformula —S—R^(k), wherein R^(k) is an alkenyl or alkynyl group,respectively.

The term “alkoxycarbonyl” includes a group having the formula—C(O)O—R^(i), wherein R^(i) is an alkyl group as defined above andwherein the total number of carbon atoms refers to the combined alkyland carbonyl moieties.

The term “acyl” includes any alkyl, alkenyl, or alkynyl wherein thecarbon at the point of attachment is substituted with an oxo group, asdefined below. The following are non-limiting examples of acyl groups:—C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl.

The term “heterocycle” includes a 5- to 7-membered monocyclic, or 7- to10-membered bicyclic, heterocyclic ring which is either saturated,unsaturated, or aromatic, and which contains from 1 or 2 heteroatomsindependently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized, including bicyclicrings in which any of the above heterocycles are fused to a benzenering. The heterocycle may be attached via any heteroatom or carbon atom.Heterocycles include, but are not limited to, heteroaryls as definedbelow, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

The term “heteroaryl” includes an aromatic 5- to 10-membered heterocyclewhich contains one, two, or more heteroatoms selected from nitrogen (N),oxygen (O), and sulfur (S). The heteroaryl can be substituted on one ormore carbon atoms with substituents such as, for example, halogen,alkyl, alkoxy, cyano, haloalkyl (e.g., trifluoromethyl), heterocyclyl(e.g., morpholinyl or pyrrolidinyl), and the like. Non-limiting examplesof heteroaryls include pyridinyl and furanyl.

The term “halogen” includes fluoro, chloro, bromo, and iodo.

The terms “optionally substituted alkyl,” “optionally substituted cyclicalkyl,” “optionally substituted alkenyl,” “optionally substitutedalkynyl,” “optionally substituted acyl,” and “optionally substitutedheterocycle” mean that, when substituted, at least one hydrogen atom isreplaced with a substituent. In the case of an “oxo” substituent (═O),two hydrogen atoms are replaced. Non-limiting examples of substituentsinclude oxo, halogen, heterocycle, —CN, —OR′, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0,1, or 2, R^(x) and R^(y) are the same or different and are independentlyhydrogen, alkyl, or heterocycle, and each of the alkyl and heterocyclesubstituents may be further substituted with one or more of oxo,halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x), and —SO_(n)NR^(x)R^(y). The term“optionally substituted,” when used before a list of substituents, meansthat each of the substituents in the list may be optionally substitutedas described herein.

In one aspect, the present invention provides a cationic lipid having astructural Formula (I):

X-A-Y—Z;  (I)

or salts thereof, wherein:

-   -   X is alkylamino;    -   A is C₁ to C₆ optionally substituted alkyl, wherein said C₁ to        C₆ optionally substituted alkyl can be saturated or unsaturated,        and wherein A may or may not be present;    -   Y is selected from the group consisting of ketal, ester,        optionally substituted carbamate, ether, and optionally        substituted amide; and    -   Z is a hydrophobic moiety consisting of three alkyl chains        wherein each of the alkyl chains has a length of from C₈ to C₁₁,        wherein each of the three alkyl chains can independently be        saturated or unsaturated, and wherein each of the three alkyl        chains is optionally substituted.

In some embodiments of the lipids of Formula (I), Z has the formula:

wherein, R₁, R₂, and R₃ are each independently selected from the groupconsisting of C₈ to C₁₁ alkyl; wherein each of R₁, R₂, and R₃ canindependently be saturated or unsaturated; and wherein each of R₁, R₂,and R₃ is optionally substituted.

In particular embodiments, a lipid of Formula (I) has one of thefollowing structures:

In some embodiments, the cationic lipid forms a salt (e.g., acrystalline salt) with one or more anions. In one particular embodiment,the cationic lipid is the oxalate (e.g., hemioxalate) salt thereof,which is preferably a crystalline salt. In particular embodiments, thecationic lipid forms a pharmaceutically acceptable salt with one or moreanions.

Also included within the scope of this invention are crystal forms,hydrates and solvates of the compounds described herein.

The compounds of the invention may be prepared by known organicsynthesis techniques, including the methods described in the Examples.In some embodiments, the synthesis of the cationic lipids of theinvention may require the use of protecting groups. Protecting groupmethodology is well known to those skilled in the art (see, e.g.,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et. al.,Wiley-Interscience, New York City, 1999). Briefly, protecting groupswithin the context of this invention are any group that reduces oreliminates the unwanted reactivity of a functional group. A protectinggroup can be added to a functional group to mask its reactivity duringcertain reactions and then removed to reveal the original functionalgroup. In certain instances, an “alcohol protecting group” is used. An“alcohol protecting group” is any group which decreases or eliminatesthe unwanted reactivity of an alcohol functional group. Protectinggroups can be added and removed using techniques well known in the art.

In certain embodiments, the cationic lipids of the present inventionhave at least one protonatable or deprotonatable group, such that thelipid is positively charged at a pH at or below physiological pH (e.g.,pH 7.4), and neutral at a second pH, preferably at or abovephysiological pH. It will be understood by one of ordinary skill in theart that the addition or removal of protons as a function of pH is anequilibrium process, and that the reference to a charged or a neutrallipid refers to the nature of the predominant species and does notrequire that all of the lipid be present in the charged or neutral form.Lipids that have more than one protonatable or deprotonatable group, orwhich are zwiterrionic, are not excluded from use in the invention.

In certain other embodiments, protonatable lipids according to theinvention have a pK_(a) of the protonatable group in the range of about4 to about 11. Most preferred is a pK_(a) of about 4 to about 7, becausethese lipids will be cationic at a lower pH formulation stage, whileparticles will be largely (though not completely) surface neutralized atphysiological pH of around pH 7.4. One of the benefits of this pK_(a) isthat at least some nucleic acid associated with the outside surface ofthe particle will lose its electrostatic interaction at physiological pHand be removed by simple dialysis, thus greatly reducing the particle'ssusceptibility to clearance.

IV. Active Agents

Active agents (e.g., therapeutic agents) include any molecule orcompound capable of exerting a desired effect on a cell, tissue, organ,or subject. Such effects may be, e.g., biological, physiological, and/orcosmetic. Active agents may be any type of molecule or compoundincluding, but not limited to, nucleic acids, peptides, polypeptides,small molecules, and mixtures thereof. Non-limiting examples of nucleicacids include interfering RNA molecules (e.g., siRNA, Dicer-substratedsRNA, shRNA, aiRNA, and/or miRNA), antisense oligonucleotides,plasmids, ribozymes, immunostimulatory oligonucleotides, and mixturesthereof. Examples of peptides or polypeptides include, withoutlimitation, antibodies (e.g., polyclonal antibodies, monoclonalantibodies, antibody fragments; humanized antibodies, recombinantantibodies, recombinant human antibodies, and/or Primatized™antibodies), cytokines, growth factors, apoptotic factors,differentiation-inducing factors, cell-surface receptors and theirligands, hormones, and mixtures thereof. Examples of small moleculesinclude, but are not limited to, small organic molecules or compoundssuch as any conventional agent or drug known to those of skill in theart.

In some embodiments, the active agent is a therapeutic agent, or a saltor derivative thereof. Therapeutic agent derivatives may betherapeutically active themselves or they may be prodrugs, which becomeactive upon further modification. Thus, in one embodiment, a therapeuticagent derivative retains some or all of the therapeutic activity ascompared to the unmodified agent, while in another embodiment, atherapeutic agent derivative is a prodrug that lacks therapeuticactivity, but becomes active upon further modification.

A. Nucleic Acids

In certain embodiments, lipid particles of the present invention areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle (e.g., LNP). In some embodiments, the nucleic acid is fullyencapsulated in the lipid particle. As used herein, the term “nucleicacid” includes any oligonucleotide or polynucleotide, with fragmentscontaining up to 60 nucleotides generally termed oligonucleotides, andlonger fragments termed polynucleotides. In particular embodiments,oligonucletoides of the invention are from about 15 to about 60nucleotides in length. Nucleic acid may be administered alone in thelipid particles of the invention, or in combination (e.g.,co-administered) with lipid particles of the invention comprisingpeptides, polypeptides, or small molecules such as conventional drugs.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally-occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also include polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake, reduced immunogenicity, and increasedstability in the presence of nucleases.

Oligonucleotides are generally classified as deoxyribooligonucleotidesor ribooligonucleotides. A deoxyribooligonucleotide consists of a5-carbon sugar called deoxyribose joined covalently to phosphate at the5′ and 3′ carbons of this sugar to form an alternating, unbranchedpolymer. A ribooligonucleotide consists of a similar repeating structurewhere the 5-carbon sugar is ribose.

The nucleic acid that is present in a nucleic acid-lipid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA are described herein and include, e.g., structuralgenes, genes including control and termination regions, andself-replicating systems such as viral or plasmid DNA. Examples ofdouble-stranded RNA are described herein and include, e.g., siRNA andother RNAi agents such as Dicer-substrate dsRNA, shRNA, aiRNA, andpre-miRNA. Single-stranded nucleic acids include, e.g., antisenseoligonucleotides, ribozymes, mature miRNA, and triplex-formingoligonucleotides.

Nucleic acids of the invention may be of various lengths, generallydependent upon the particular form of nucleic acid. For example, inparticular embodiments, plasmids or genes may be from about 1,000 toabout 100,000 nucleotide residues in length. In particular embodiments,oligonucleotides may range from about 10 to about 100 nucleotides inlength. In various related embodiments, oligonucleotides, bothsingle-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 60 nucleotides, from about 15 to about 60nucleotides, from about 20 to about 50 nucleotides, from about 15 toabout 30 nucleotides, or from about 20 to about 30 nucleotides inlength.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe invention specifically hybridizes to or is complementary to a targetpolynucleotide sequence. The terms “specifically hybridizable” and“complementary” as used herein indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. In preferred embodiments,an oligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target sequence interferes with the normalfunction of the target sequence to cause a loss of utility or expressiontherefrom, and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or, in the case of in vitro assays, under conditions in whichthe assays are conducted. Thus, the oligonucleotide may include 1, 2, 3,or more base substitutions as compared to the region of a gene or mRNAsequence that it is targeting or to which it specifically hybridizes.

1. siRNA

The siRNA component of the nucleic acid-lipid particles of the presentinvention is capable of silencing the expression of a target gene ofinterest. Each strand of the siRNA duplex is typically about 15 to about60 nucleotides in length, preferably about 15 to about 30 nucleotides inlength. In certain embodiments, the siRNA comprises at least onemodified nucleotide. The modified siRNA is generally lessimmunostimulatory than a corresponding unmodified siRNA sequence andretains RNAi activity against the target gene of interest. In someembodiments, the modified siRNA contains at least one 2′OMe purine orpyrimidine nucleotide such as a 2′OMe-guanosine, 2′OMe-uridine,2′OMe-adenosine, and/or 2′OMe-cytosine nucleotide. The modifiednucleotides can be present in one strand (i.e., sense or antisense) orboth strands of the siRNA. In some preferred embodiments, one or more ofthe uridine and/or guanosine nucleotides are modified (e.g.,2′OMe-modified) in one strand (i.e., sense or antisense) or both strandsof the siRNA. In these embodiments, the modified siRNA can furthercomprise one or more modified (e.g., 2′OMe-modified) adenosine and/ormodified (e.g., 2′OMe-modified) cytosine nucleotides. In other preferredembodiments, only uridine and/or guanosine nucleotides are modified(e.g., 2′OMe-modified) in one strand (i.e., sense or antisense) or bothstrands of the siRNA. The siRNA sequences may have overhangs (e.g., 3′or 5′ overhangs as described in Elbashir et al., Genes Dev., 15:188(2001) or Nykänen et al., Cell, 107:309 (2001)), or may lack overhangs(i.e., have blunt ends).

In particular embodiments, the selective incorporation of modifiednucleotides such as 2′OMe uridine and/or guanosine nucleotides into thedouble-stranded region of either or both strands of the siRNA reduces orcompletely abrogates the immune response to that siRNA molecule. Incertain instances, the immunostimulatory properties of specific siRNAsequences and their ability to silence gene expression can be balancedor optimized by the introduction of minimal and selective 2′OMemodifications within the double-stranded region of the siRNA duplex.This can be achieved at therapeutically viable siRNA doses withoutcytokine induction, toxicity, and off-target effects associated with theuse of unmodified siRNA.

The modified siRNA generally comprises from about 1% to about 100%(e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) modified nucleotides in the double-stranded region ofthe siRNA duplex. In certain embodiments, one, two, three, four, five,six, seven, eight, nine, ten, or more of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides. Incertain other embodiments, some or all of the modified nucleotides inthe double-stranded region of the siRNA are 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more nucleotides apart from each other. In one preferredembodiment, none of the modified nucleotides in the double-strandedregion of the siRNA are adjacent to each other (e.g., there is a gap ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides betweeneach modified nucleotide).

In some embodiments, less than about 50% (e.g., less than about 49%,48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, or 36%,preferably less than about 35%, 34%, 33%, 32%, 31%, or 30%) of thenucleotides in the double-stranded region of the siRNA comprise modified(e.g., 2′OMe) nucleotides. In one aspect of these embodiments, less thanabout 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, lessthan about 50% of the nucleotides in the double-stranded region of thesiRNA comprise 2′OMe nucleotides, wherein the siRNA comprises 2′OMenucleotides in both strands of the siRNA, wherein the siRNA comprises atleast one 2′OMe-guanosine nucleotide and at least one 2′OMe-uridinenucleotide, and wherein the siRNA does not comprise 2′OMe-cytosinenucleotides in the double-stranded region. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the 2′OMe nucleotides in the double-stranded region are notadjacent to each other.

In other embodiments, from about 1% to about 50% (e.g., from about5%-50%, 10%-50%, 15%-50%, 20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%,45%-50%, 5%-45%, 10%-45%, 15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%,40%-45%, 5%-40%, 10%-40%, 15%-40%, 20%-40%, 25%-40%, 25%-39%, 25%-38%,25%-37%, 25%-36%, 26%-39%, 26%-38%, 26%-37%, 26%-36%, 27%-39%, 27%-38%,27%-37%, 27%-36%, 28%-39%, 28%-38%, 28%-37%, 28%-36%, 29%-39%, 29%-38%,29%-37%, 29%-36%, 30%-40%, 30%-39%, 30%-38%, 30%-37%, 30%-36%, 31%-39%,31%-38%, 31%-37%, 31%-36%, 32%-39%, 32%-38%, 32%-37%, 32%-36%, 33%-39%,33%-38%, 33%-37%, 33%-36%, 34%-39%, 34%-38%, 34%-37%, 34%-36%, 35%-40%,5%-35%, 10%-35%, 15%-35%, 20%-35%, 21%-35%, 22%-35%, 23%-35%, 24%-35%,25%-35%, 26%-35%, 27%-35%, 28%-35%, 29%-35%, 30%-35%, 31%-35%, 32%-35%,33%-35%, 34%-35%, 30%-34%, 31%-34%, 32%-34%, 33%-34%, 30%-33%, 31%-33%,32%-33%, 30%-32%, 31%-32%, 25%-34%, 25%-33%, 25%-32%, 25%-31%, 26%-34%,26%-33%, 26%-32%, 26%-31%, 27%-34%, 27%-33%, 27%-32%, 27%-31%, 28%-34%,28%-33%, 28%-32%, 28%-31%, 29%-34%, 29%-33%, 29%-32%, 29%-31%, 5%-30%,10%-30%, 15%-30%, 20%-34%, 20%-33%, 20%-32%, 20%-31%, 20%-30%, 21%-30%,22%-30%, 23%-30%, 24%-30%, 25%-30%, 25%-29%, 25%-28%, 25%-27%, 25%-26%,26%-30%, 26%-29%, 26%-28%, 26%-27%, 27%-30%, 27%-29%, 27%-28%, 28%-30%,28%-29%, 29%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-29%, 20%-28%, 20%-27%,20%-26%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%, 10%-15%, or 5%-10%)of the nucleotides in the double-stranded region of the siRNA comprisemodified nucleotides. In one aspect of these embodiments, from about 1%to about 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, fromabout 1% to about 50% of the nucleotides in the double-stranded regionof the siRNA comprise 2′OMe nucleotides, wherein the siRNA comprises2′OMe nucleotides in both strands of the siRNA, wherein the siRNAcomprises at least one 2′OMe-guanosine nucleotide and at least one2′OMe-uridine nucleotide, and wherein the siRNA does not comprise2′OMe-cytosine nucleotides in the double-stranded region. In anotheraspect of these embodiments, from about 1% to about 50% of thenucleotides in the double-stranded region of the siRNA comprise 2′OMenucleotides, wherein the siRNA comprises 2′OMe nucleotides in bothstrands of the modified siRNA, wherein the siRNA comprises 2′OMenucleotides selected from the group consisting of 2′OMe-guanosinenucleotides, 2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, andmixtures thereof, and wherein the 2′OMe nucleotides in thedouble-stranded region are not adjacent to each other.

Additional ranges, percentages, and patterns of modifications that maybe introduced into siRNA are described in U.S. Patent ApplicationPublication No. 2007/0135372, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

a) Selection of siRNA Sequences

Suitable siRNA sequences can be identified using any means known in theart. Typically, the methods described in Elbashir et al., Nature,411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) arecombined with rational design rules set forth in Reynolds et al., NatureBiotech., 22 (3):326-330 (2004).

As a non-limiting example, the nucleotide sequence 3′ of the AUG startcodon of a transcript from the target gene of interest may be scannedfor dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, wherein N=C, G,or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). Thenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences (i.e., a target sequence or a sense strandsequence). Typically, the 19, 21, 23, 25, 27, 29, 31, 33, 35, or morenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences. In some embodiments, the dinucleotidesequence is an AA or NA sequence and the 19 nucleotides immediately 3′to the AA or NA dinucleotide are identified as potential siRNAsequences. siRNA sequences are usually spaced at different positionsalong the length of the target gene. To further enhance silencingefficiency of the siRNA sequences, potential siRNA sequences may beanalyzed to identify sites that do not contain regions of homology toother coding sequences, e.g., in the target cell or organism. Forexample, a suitable siRNA sequence of about 21 base pairs typically willnot have more than 16-17 contiguous base pairs of homology to codingsequences in the target cell or organism. If the siRNA sequences are tobe expressed from an RNA Pol III promoter, siRNA sequences lacking morethan 4 contiguous A's or T's are selected.

Once a potential siRNA sequence has been identified, a complementarysequence (i.e., an antisense strand sequence) can be designed. Apotential siRNA sequence can also be analyzed using a variety ofcriteria known in the art. For example, to enhance their silencingefficiency, the siRNA sequences may be analyzed by a rational designalgorithm to identify sequences that have one or more of the followingfeatures: (1) G/C content of about 25% to about 60% G/C; (2) at least 3A/Us at positions 15-19 of the sense strand; (3) no internal repeats;(4) an A at position 19 of the sense strand; (5) an A at position 3 ofthe sense strand; (6) a U at position 10 of the sense strand; (7) no G/Cat position 19 of the sense strand; and (8) no G at position 13 of thesense strand. siRNA design tools that incorporate algorithms that assignsuitable values of each of these features and are useful for selectionof siRNA can be found at, e.g.,http://ihome.ust.h5/˜bokcmho/siRNA/siRNA.html. One of skill in the artwill appreciate that sequences with one or more of the foregoingcharacteristics may be selected for further analysis and testing aspotential siRNA sequences.

Additionally, potential siRNA sequences with one or more of thefollowing criteria can often be eliminated as siRNA: (1) sequencescomprising a stretch of 4 or more of the same base in a row; (2)sequences comprising homopolymers of Gs (i.e., to reduce possiblenon-specific effects due to structural characteristics of thesepolymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC,AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in arow; and (5) sequences comprising direct repeats of 4 or more baseswithin the candidates resulting in internal fold-back structures.However, one of skill in the art will appreciate that sequences with oneor more of the foregoing characteristics may still be selected forfurther analysis and testing as potential siRNA sequences.

In some embodiments, potential siRNA sequences may be further analyzedbased on siRNA duplex asymmetry as described in, e.g., Khvorova et al.,Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003).In other embodiments, potential siRNA sequences may be further analyzedbased on secondary structure at the target site as described in, e.g.,Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example,secondary structure at the target site can be modeled using the Mfoldalgorithm (available at http://mfold.burnetedu.au/rnaform) to selectsiRNA sequences which favor accessibility at the target site where lesssecondary structure in the form of base-pairing and stem-loops ispresent.

Once a potential siRNA sequence has been identified, the sequence can beanalyzed for the presence of any immunostimulatory properties, e.g.,using an in vitro cytokine assay or an in vivo animal model. Motifs inthe sense and/or antisense strand of the siRNA sequence such as GU-richmotifs (e.g., 5′-GU-3′, 5′-UGU-3′, 5′-GUGU-3′, 5′-UGUGU-3′, etc.) canalso provide an indication of whether the sequence may beimmunostimulatory. Once an siRNA molecule is found to beimmunostimulatory, it can then be modified to decrease itsimmunostimulatory properties as described herein. As a non-limitingexample, an siRNA sequence can be contacted with a mammalian respondercell under conditions such that the cell produces a detectable immuneresponse to determine whether the siRNA is an immunostimulatory or anon-immunostimulatory siRNA. The mammalian responder cell may be from anaïve mammal (i.e., a mammal that has not previously been in contactwith the gene product of the siRNA sequence). The mammalian respondercell may be, e.g., a peripheral blood mononuclear cell (PBMC), amacrophage, and the like. The detectable immune response may compriseproduction of a cytokine or growth factor such as, e.g., TNF-α, IFN-α,IFN-β, IFN-γ, IL-6, IL-12, or a combination thereof Δn siRNA moleculeidentified as being immunostimulatory can then be modified to decreaseits immunostimulatory properties by replacing at least one of thenucleotides on the sense and/or antisense strand with modifiednucleotides. For example, less than about 30% (e.g., less than about30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in thedouble-stranded region of the siRNA duplex can be replaced with modifiednucleotides such as 2′OMe nucleotides. The modified siRNA can then becontacted with a mammalian responder cell as described above to confirmthat its immunostimulatory properties have been reduced or abrogated.

Suitable in vitro assays for detecting an immune response include, butare not limited to, the double monoclonal antibody sandwich immunoassaytechnique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh (1970)); the “Western blot” method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays(ELISA) as described, for example, by Raines et al., J. Biol. Chem.,257:5154-5160 (1982); immunocytochemical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980));and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad.Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassaysdescribed above, a number of other immunoassays are available, includingthose described in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Thedisclosures of these references are herein incorporated by reference intheir entirety for all purposes.

A non-limiting example of an in vivo model for detecting an immuneresponse includes an in vivo mouse cytokine induction assay as describedin, e.g., Judge et al., Mol. Ther., 13:494-505 (2006). In certainembodiments, the assay that can be performed as follows: (1) siRNA canbe administered by standard intravenous injection in the lateral tailvein; (2) blood can be collected by cardiac puncture about 6 hours afteradministration and processed as plasma for cytokine analysis; and (3)cytokines can be quantified using sandwich ELISA kits according to themanufacturer's instructions (e.g., mouse and human IFN-α (PBLBiomedical; Piscataway, N.J.); human IL-6 and TNF-α (eBioscience; SanDiego, Calif.); and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; SanDiego, Calif.)).

Monoclonal antibodies that specifically bind cytokines and growthfactors are commercially available from multiple sources and can begenerated using methods known in the art (see, e.g., Kohler et al.,Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)).Generation of monoclonal antibodies has been previously described andcan be accomplished by any means known in the art (Buhring et al., inHybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, themonoclonal antibody is labeled (e.g., with any composition detectable byspectroscopic, photochemical, biochemical, electrical, optical, orchemical means) to facilitate detection.

b) Generating siRNA Molecules

siRNA can be provided in several forms including, e.g., as one or moreisolated small-interfering RNA (siRNA) duplexes, as longerdouble-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from atranscriptional cassette in a DNA plasmid. In some embodiments, siRNAmay be produced enzymatically or by partial/total organic synthesis, andmodified ribonucleotides can be introduced by in vitro enzymatic ororganic synthesis. In certain instances, each strand is preparedchemically. Methods of synthesizing RNA molecules are known in the art,e.g., the chemical synthesis methods as described in Verma and Eckstein(1998) or as described herein.

An RNA population can be used to provide long precursor RNAs, or longprecursor RNAs that have substantial or complete identity to a selectedtarget sequence can be used to make the siRNA. The RNAs can be isolatedfrom cells or tissue, synthesized, and/or cloned according to methodswell known to those of skill in the art. The RNA can be a mixedpopulation (obtained from cells or tissue, transcribed from cDNA,subtracted, selected, etc.), or can represent a single target sequence.RNA can be naturally occurring (e.g., isolated from tissue or cellsamples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCRproducts or a cloned cDNA), or chemically synthesized.

To form a long dsRNA, for synthetic RNAs, the complement is alsotranscribed in vitro and hybridized to form a dsRNA. If a naturallyoccurring RNA population is used, the RNA complements are also provided(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,by transcribing cDNAs corresponding to the RNA population, or by usingRNA polymerases. The precursor RNAs are then hybridized to form doublestranded RNAs for digestion. The dsRNAs can be directly administered toa subject or can be digested in vitro prior to administration.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see,U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994). The disclosures of these references are herein incorporatedby reference in their entirety for all purposes.

Preferably, siRNA are chemically synthesized. The oligonucleotides thatcomprise the siRNA molecules of the invention can be synthesized usingany of a variety of techniques known in the art, such as those describedin Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al.,Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res.,23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59(1997). The synthesis of oligonucleotides makes use of common nucleicacid protecting and coupling groups, such as dimethoxytrityl at the5′-end and phosphoramidites at the 3′-end. As a non-limiting example,small scale syntheses can be conducted on an Applied Biosystemssynthesizer using a 0.2 μmol scale protocol. Alternatively, syntheses atthe 0.2 μmol scale can be performed on a 96-well plate synthesizer fromProtogene (Palo Alto, Calif.). However, a larger or smaller scale ofsynthesis is also within the scope of this invention. Suitable reagentsfor oligonucleotide synthesis, methods for RNA deprotection, and methodsfor RNA purification are known to those of skill in the art.

siRNA molecules can also be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousoligonucleotide fragment or strand separated by a cleavable linker thatis subsequently cleaved to provide separate fragments or strands thathybridize to form the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker. The tandem synthesis of siRNA can bereadily adapted to both multiwell/multiplate synthesis platforms as wellas large scale synthesis platforms employing batch reactors, synthesiscolumns, and the like. Alternatively, siRNA molecules can be assembledfrom two distinct oligonucleotides, wherein one oligonucleotidecomprises the sense strand and the other comprises the antisense strandof the siRNA. For example, each strand can be synthesized separately andjoined together by hybridization or ligation following synthesis and/ordeprotection. In certain other instances, siRNA molecules can besynthesized as a single continuous oligonucleotide fragment, where theself-complementary sense and antisense regions hybridize to form ansiRNA duplex having hairpin secondary structure.

c) Modifying siRNA Sequences

In certain aspects, siRNA molecules comprise a duplex having two strandsand at least one modified nucleotide in the double-stranded region,wherein each strand is about 15 to about 60 nucleotides in length.Advantageously, the modified siRNA is less immunostimulatory than acorresponding unmodified siRNA sequence, but retains the capability ofsilencing the expression of a target sequence. In preferred embodiments,the degree of chemical modifications introduced into the siRNA moleculestrikes a balance between reduction or abrogation of theimmunostimulatory properties of the siRNA and retention of RNAiactivity. As a non-limiting example, an siRNA molecule that targets agene of interest can be minimally modified (e.g., less than about 30%,25%, 20%, 15%, 10%, or 5% modified) at selective uridine and/orguanosine nucleotides within the siRNA duplex to eliminate the immuneresponse generated by the siRNA while retaining its capability tosilence target gene expression.

Examples of modified nucleotides suitable for use in the inventioninclude, but are not limited to, ribonucleotides having a 2′-O-methyl(2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a Northern conformation such as thosedescribed in, e.g., Saenger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), are also suitable for use in siRNAmolecules. Such modified nucleotides include, without limitation, lockednucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl)(MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro (2′Cl) nucleotides, and 2′-azidonucleotides. In certain instances, the siRNA molecules described hereininclude one or more G-clamp nucleotides. A G-clamp nucleotide refers toa modified cytosine analog wherein the modifications confer the abilityto hydrogen bond both Watson-Crick and Hoogsteen faces of acomplementary guanine nucleotide within a duplex (see, e.g., Lin et al.,J. Am. Chem. Soc., 120:8531-8532 (1998)). In addition, nucleotideshaving a nucleotide base analog such as, for example, C-phenyl,C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res.,29:2437-2447 (2001)) can be incorporated into siRNA molecules.

In certain embodiments, siRNA molecules may further comprise one or morechemical modifications such as terminal cap moieties, phosphate backbonemodifications, and the like. Examples of terminal cap moieties include,without limitation, inverted deoxy abasic residues, glycerylmodifications, 4′,5′-methylene nucleotides, 1-(β-D-erythrofuranosyl)nucleotides, 4′-thio nucleotides, carbocyclic nucleotides,1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modifiedbase nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties,3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties,3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties,5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties,5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropylphosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate,hydroxypropyl phosphate, 1,4-butanediol phosphate, 3′-phosphoramidate,5′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron49:1925 (1993)). Non-limiting examples of phosphate backbonemodifications (i.e., resulting in modified internucleotide linkages)include phosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyimide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker etal., Nucleic Acid Analogues: Synthesis and Properties, in ModernSynthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39 (1994)). Such chemical modifications canoccur at the 5′-end and/or 3′-end of the sense strand, antisense strand,or both strands of the siRNA. The disclosures of these references areherein incorporated by reference in their entirety for all purposes.

In some embodiments, the sense and/or antisense strand of the siRNAmolecule can further comprise a 3′-terminal overhang having about 1 toabout 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides, modified (e.g.,2′OMe) and/or unmodified uridine ribonucleotides, and/or any othercombination of modified (e.g., 2′OMe) and unmodified nucleotides.

Additional examples of modified nucleotides and types of chemicalmodifications that can be introduced into siRNA molecules are described,e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent ApplicationPublication Nos. 2004/0192626, 2005/0282188, and 2007/0135372, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

The siRNA molecules described herein can optionally comprise one or morenon-nucleotides in one or both strands of the siRNA. As used herein, theterm “non-nucleotide” refers to any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including sugar and/or phosphate substitutions, andallows the remaining bases to exhibit their activity. The group orcompound is abasic in that it does not contain a commonly recognizednucleotide base such as adenosine, guanine, cytosine, uracil, or thymineand therefore lacks a base at the 1′-position.

In other embodiments, chemical modification of the siRNA comprisesattaching a conjugate to the siRNA molecule. The conjugate can beattached at the 5′ and/or 3′-end of the sense and/or antisense strand ofthe siRNA via a covalent attachment such as, e.g., a biodegradablelinker. The conjugate can also be attached to the siRNA, e.g., through acarbamate group or other linking group (see, e.g., U.S. PatentApplication Publication Nos. 2005/0074771, 2005/0043219, and2005/0158727). In certain instances, the conjugate is a molecule thatfacilitates the delivery of the siRNA into a cell. Examples of conjugatemolecules suitable for attachment to siRNA include, without limitation,steroids such as cholesterol, glycols such as polyethylene glycol (PEG),human serum albumin (HSA), fatty acids, carotenoids, terpenes, bileacids, folates (e.g., folic acid, folate analogs and derivativesthereof), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids,peptides, ligands for cellular receptors capable of mediating cellularuptake, and combinations thereof (see, e.g., U.S. Patent ApplicationPublication Nos. 2003/0130186, 2004/0110296, and 2004/0249178; U.S. Pat.No. 6,753,423). Other examples include the lipophilic moiety, vitamin,polymer, peptide, protein, nucleic acid, small molecule,oligosaccharide, carbohydrate cluster, intercalator, minor groovebinder, cleaving agent, and cross-linking agent conjugate moleculesdescribed in U.S. Patent Application Publication Nos. 2005/0119470 and2005/0107325. Yet other examples include the 2′-O-alkyl amine,2′-O-alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine,cationic peptide, guanidinium group, amidininium group, cationic aminoacid conjugate molecules described in U.S. Patent ApplicationPublication No. 2005/0153337. Additional examples include thehydrophobic group, membrane active compound, cell penetrating compound,cell targeting signal, interaction modifier, and steric stabilizerconjugate molecules described in U.S. Patent Application Publication No.2004/0167090. Further examples include the conjugate molecules describedin U.S. Patent Application Publication No. 2005/0239739. The type ofconjugate used and the extent of conjugation to the siRNA molecule canbe evaluated for improved pharmacokinetic profiles, bioavailability,and/or stability of the siRNA while retaining RNAi activity. As such,one skilled in the art can screen siRNA molecules having variousconjugates attached thereto to identify ones having improved propertiesand full RNAi activity using any of a variety of well-known in vitrocell culture or in vivo animal models. The disclosures of theabove-described patent documents are herein incorporated by reference intheir entirety for all purposes.

d) Target Genes

The siRNA component of the nucleic acid-lipid particles described hereincan be used to downregulate or silence the translation (i.e.,expression) of a gene of interest. Genes of interest include, but arenot limited to, genes associated with viral infection and survival,genes associated with metabolic diseases and disorders (e.g., liverdiseases and disorders), genes associated with tumorigenesis or celltransformation (e.g., cancer), angiogenic genes, immunomodulator genessuch as those associated with inflammatory and autoimmune responses,receptor ligand genes, and genes associated with neurodegenerativedisorders.

In particular embodiments, the present invention provides a cocktail oftwo, three, four, five, six, seven, eight, nine, ten, or more siRNAmolecules that silences the expression of multiple genes of interest. Insome embodiments, the cocktail of siRNA molecules is fully encapsulatedin a lipid particle such as a nucleic acid-lipid particle (e.g., LNP).The siRNA molecules may be co-encapsulated in the same lipid particle,or each siRNA species present in the cocktail may be formulated inseparate particles.

Genes associated with viral infection and survival include thoseexpressed by a host (e.g., a host factor such as tissue factor (TF)) ora virus in order to bind, enter, and replicate in a cell. Of particularinterest are viral sequences associated with chronic viral diseases.Viral sequences of particular interest include sequences of Filovirusessuch as Ebola virus and Marburg virus (see, e.g., Geisbert et al., JInfect. Dis., 193:1650-1657 (2006)); Arenaviruses such as Lassa virus,Junin virus, Machupo virus, Guanarito virus, and Sabia virus (Buchmeieret al., Arenaviridae: the viruses and their replication, In: FIELDSVIROLOGY, Knipe et al. (eds.), 4th ed., Lippincott-Raven, Philadelphia,(2001)); Influenza viruses such as Influenza A, B, and C viruses, (see,e.g., Steinhauer et al., Annu Rev Genet., 36:305-332 (2002); and Neumannet al., J Gen Virol., 83:2635-2662 (2002)); Hepatitis viruses (see,e.g., Hamasaki et al., FEBS Lett., 543:51 (2003); Yokota et al., EMBORep., 4:602 (2003); Schlomai et al., Hepatology, 37:764 (2003); Wilsonet al., Proc. Natl. Acad. Sci. USA, 100:2783 (2003); Kapadia et al.,Proc. Natl. Acad. Sci. USA, 100:2014 (2003); and FIELDS VIROLOGY, Knipeet al. (eds.), 4th ed., Lippincott-Raven, Philadelphia (2001)); HumanImmunodeficiency Virus (HIV) (Banerjea et al., Mol. Ther., 8:62 (2003);Song et al., J. Virol., 77:7174 (2003); Stephenson, JAMA, 289:1494(2003); Qin et al., Proc. Natl. Acad. Sci. USA, 100:183 (2003)); Herpesviruses (Jia et al., J. Virol., 77:3301 (2003)); and Human PapillomaViruses (HPV) (Hall et al., J. Virol., 77:6066 (2003); Jiang et al.,Oncogene, 21:6041 (2002)).

Exemplary Filovirus nucleic acid sequences that can be silenced include,but are not limited to, nucleic acid sequences encoding structuralproteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein(L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein(GP), VP24). Complete genome sequences for Ebola virus are set forth in,e.g., Genbank Accession Nos. NC_002549; AY769362; NC_006432; NC_004161;AY729654; AY354458; AY142960; AB050936; AF522874; AF499101; AF272001;and AF086833. Ebola virus VP24 sequences are set forth in, e.g., GenbankAccession Nos. U77385 and AY058897. Ebola virus L-pol sequences are setforth in, e.g., Genbank Accession No. X67110. Ebola virus VP40 sequencesare set forth in, e.g., Genbank Accession No. AY058896. Ebola virus NPsequences are set forth in, e.g., Genbank Accession No. AY058895. Ebolavirus GP sequences are set forth in, e.g., Genbank Accession No.AY058898; Sanchez et al., Virus Res., 29:215-240 (1993); Will et al., J.Virol., 67:1203-1210 (1993); Volchkov et al., FEBS Lett., 305:181-184(1992); and U.S. Pat. No. 6,713,069. Additional Ebola virus sequencesare set forth in, e.g., Genbank Accession Nos. L11365 and X61274.Complete genome sequences for Marburg virus are set forth in, e.g.,Genbank Accession Nos. NC_001608; AY430365; AY430366; and AY358025.Marburg virus GP sequences are set forth in, e.g., Genbank AccessionNos. AF005734; AF005733; and AF005732. Marburg virus VP35 sequences areset forth in, e.g., Genbank Accession Nos. AF005731 and AF005730.Additional Marburg virus sequences are set forth in, e.g., GenbankAccession Nos. X64406; Z29337; AF005735; and Z12132. Non-limitingexamples of siRNA molecules targeting Ebola virus and Marburg virusnucleic acid sequences include those described in U.S. PatentApplication Publication No. 2007/0135370 and U.S. ProvisionalApplication No. 61/286,741, filed Dec. 15, 2009, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

Exemplary Arenavirus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences encodingnucleoprotein (NP), glycoprotein (GP), L-polymerase (L), and Z protein(Z). Complete genome sequences for Lassa virus are set forth in, e.g.,Genbank Accession Nos. NC_004296 (LASV segment S) and NC_004297 (LASVsegment L). Non-limiting examples of siRNA molecules targeting Lassavirus nucleic acid sequences include those described in U.S. ProvisionalApplication No. 61/319,855, filed Mar. 31, 2010, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

Exemplary host nucleic acid sequences that can be silenced include, butare not limited to, nucleic acid sequences encoding host factors such astissue factor (TF) that are known to play a role in the pathogenisis ofhemorrhagic fever viruses. The mRNA sequence of TF is set forth inGenbank Accession No. NM_001993. Those of skill in the art willappreciate that TF is also known as F3, coagulation factor III,thromboplastin, and CD142. Non-limiting examples of siRNA moleculestargeting TF nucleic acid sequences include those described in U.S.Provisional Application No. 61/319,855, filed Mar. 31, 2010, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

Exemplary Influenza virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences encodingnucleoprotein (NP), matrix proteins (M1 and M2), nonstructural proteins(NS1 and NS2), RNA polymerase (PA, PB1, PB2), neuraminidase (NA), andhaemagglutinin (HA). Influenza A NP sequences are set forth in, e.g.,Genbank Accession Nos. NC_004522; AY818138; AB166863; AB188817;AB189046; AB189054; AB189062; AY646169; AY646177; AY651486; AY651493;AY651494; AY651495; AY651496; AY651497; AY651498; AY651499; AY651500;AY651501; AY651502; AY651503; AY651504; AY651505; AY651506; AY651507;AY651509; AY651528; AY770996; AY790308; AY818138; and AY818140.Influenza A PA sequences are set forth in, e.g., Genbank Accession Nos.AY818132; AY790280; AY646171; AY818132; AY818133; AY646179; AY818134;AY551934; AY651613; AY651610; AY651620; AY651617; AY651600; AY651611;AY651606; AY651618; AY651608; AY651607; AY651605; AY651609; AY651615;AY651616; AY651640; AY651614; AY651612; AY651621; AY651619; AY770995;and AY724786. Non-limiting examples of siRNA molecules targetingInfluenza virus nucleic acid sequences include those described in U.S.Patent Application Publication No. 2007/0218122, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

Exemplary hepatitis virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences involved intranscription and translation (e.g., En1, En2, X, P) and nucleic acidsequences encoding structural proteins (e.g., core proteins including Cand C-related proteins, capsid and envelope proteins including S, M,and/or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY,supra). Exemplary Hepatits C virus (HCV) nucleic acid sequences that canbe silenced include, but are not limited to, the 5′-untranslated region(5′-UTR), the 3′-untranslated region (3′-UTR), the polyproteintranslation initiation codon region, the internal ribosome entry site(IRES) sequence, and/or nucleic acid sequences encoding the coreprotein, the E1 protein, the E2 protein, the p7 protein, the NS2protein, the NS3 protease/helicase, the NS4A protein, the NS4B protein,the NS5A protein, and/or the NS5B RNA-dependent RNA polymerase. HCVgenome sequences are set forth in, e.g., Genbank Accession Nos.NC_004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), NC_009823 (HCVgenotype 2), NC_009824 (HCV genotype 3), NC_009825 (HCV genotype 4),NC_009826 (HCV genotype 5), and NC_009827 (HCV genotype 6). Hepatitis Avirus nucleic acid sequences are set forth in, e.g., Genbank AccessionNo. NC_001489; Hepatitis B virus nucleic acid sequences are set forthin, e.g., Genbank Accession No. NC_003977; Hepatitis D virus nucleicacid sequence are set forth in, e.g., Genbank Accession No. NC_001653;Hepatitis E virus nucleic acid sequences are set forth in, e.g., GenbankAccession No. NC_001434; and Hepatitis G virus nucleic acid sequencesare set forth in, e.g., Genbank Accession No. NC_001710. Silencing ofsequences that encode genes associated with viral infection and survivalcan conveniently be used in combination with the administration ofconventional agents used to treat the viral condition. Non-limitingexamples of siRNA molecules targeting hepatitis virus nucleic acidsequences include those described in U.S. Patent Application PublicationNos. 2006/0281175, 2005/0058982, and 2007/0149470; U.S. Pat. No.7,348,314; and PCT Application No. PCT/CA2010/000444, entitled“Compositions and Methods for Silencing Hepatitis C Virus Expression,”filed Mar. 19, 2010, bearing Attorney Docket No. 020801-008910PC, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

Genes associated with metabolic diseases and disorders (e.g., disordersin which the liver is the target and liver diseases and disorders)include, but are not limited to, genes expressed in dyslipidemia, suchas, e.g., apolipoprotein B (APOB) (Genbank Accession No. NM_000384),apolipoprotein CIII (APOC3) (Genbank Accession Nos. NM_000040 andNG_008949 REGION: 5001 . . . 8164), apolipoprotein E (APOE) (GenbankAccession Nos. NM_000041 and NG 007084 REGION: 5001 . . . 8612),proprotein convertase subtilisin/kexin type 9 (PCSK9) (Genbank AccessionNo. NM_174936), diacylglycerol O-acyltransferase type 1 (DGAT1) (GenbankAccession No. NM_012079), diacylglyerol O-acyltransferase type 2 (DGAT2)(Genbank Accession No. NM_032564), liver X receptors such as LXRα andLXRβ (Genback Accession No. NM_007121), farnesoid X receptors (FXR)(Genbank Accession No. NM_005123), sterol-regulatory element bindingprotein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutarylcoenzyme-A reductase (HMG coenzyme-A reductase); and genes expressed indiabetes, such as, e.g., glucose 6-phosphatase (see, e.g., Forman etal., Cell, 81:687 (1995); Seol et al., Mol. Endocrinol., 9:72 (1995),Zavacki et al., Proc. Natl. Acad. Sci. USA, 94:7909 (1997); Sakai etal., Cell, 85:1037-1046 (1996); Duncan et al., J. Biol. Chem.,272:12778-12785 (1997); Willy et al., Genes Dev., 9:1033-1045 (1995);Lehmann et al., J. Biol. Chem., 272:3137-3140 (1997); Janowski et al.,Nature, 383:728-731 (1996); and Peet et al., Cell, 93:693-704 (1998)).

One of skill in the art will appreciate that genes associated withmetabolic diseases and disorders (e.g., diseases and disorders in whichthe liver is a target and liver diseases and disorders) include genesthat are expressed in the liver itself as well as and genes expressed inother organs and tissues. Silencing of sequences that encode genesassociated with metabolic diseases and disorders can conveniently beused in combination with the administration of conventional agents usedto treat the disease or disorder. Non-limiting examples of siRNAmolecules targeting the APOB gene include those described in U.S. PatentApplication Publication Nos. 2006/0134189, 2006/0105976, and2007/0135372, and PCT Publication No. WO 04/091515, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. Non-limiting examples of siRNA molecules targeting the APOC3gene include those described in PCT Application No. PCT/CA2010/000120,filed Jan. 26, 2010, the disclosure of which is herein incorporated byreference in its entirety for all purposes. Non-limiting examples ofsiRNA molecules targeting the PCSK9 gene include those described in U.S.Patent Application Publication Nos. 2007/0173473, 2008/0113930, and2008/0306015, the disclosures of which are herein incorporated byreference in their entirety for all purposes. Exemplary siRNA moleculestargeting the DGAT1 gene may be designed using the antisense compoundsdescribed in U.S. Patent Application Publication No. 2004/0185559, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Exemplary siRNA molecules targeting the DGAT2 gene maybe designed using the antisense compounds described in U.S. PatentApplication Publication No. 2005/0043524, the disclosure of which isherein incorporated by reference in its entirety for all purposes.

Genes associated with tumorigenesis or cell transformation (e.g., canceror other neoplasia) include, for example, genes involved in p53ubiquitination, c-Jun ubiquitination, histone deacetylation, cell cycleregulation, transcriptional regulation, and combinations thereof.Non-limiting examples of gene sequences associated with tumorigenesis orcell transformation include serine/threonine kinases such as polo-likekinase 1 (PLK-1) (Genbank Accession No. NM 005030; Barr et al., Nat.Rev. Mol. Cell Biol., 5:429-440 (2004)) and cyclin-dependent kinase 4(CDK4) (Genbank Accession No. NM_000075); ubiquitin ligases such as COP1(RFWD2; Genbank Accession Nos. NM_022457 and NM_001001740) and ring-box1 (RBX1) (ROC1; Genbank Accession No. NM 014248); tyrosine kinases suchas WEE1 (Genbank Accession Nos. NM_003390 and NM_001143976); mitotickinesins such as Eg5 (KSP, KIF11; Genbank Accession No. NM_004523);transcription factors such as forkhead box M1 (FOXM1) (Genbank AccessionNos. NM_202002, NM_021953, and NM_202003) and RAM2 (R1 or CDCA7L;Genbank Accession Nos. NM_018719, NM_001127370, and NM 001127371);inhibitors of apoptosis such as XIAP (Genbank Accession No. NM_001167);COP9 signalosome subunits such as CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1;Genbank Accession No. NM_006837); CSN6, CSN7A, CSN7B, and CSN8; andhistone deacetylases such as HDAC1, HDAC2 (Genbank Accession No.NM_001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc.

Non-limiting examples of siRNA molecules targeting the PLK-1 geneinclude those described in U.S. Patent Application Publication Nos.2005/0107316 and 2007/0265438; and PCT Publication No. WO 09/082817, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes. Non-limiting examples of siRNA moleculestargeting the Eg5 and XIAP genes include those described in U.S. PatentApplication Publication No. 2009/0149403, the disclosure of which isherein incorporated by reference in its entirety for all purposes.Non-limiting examples of siRNA molecules targeting the CSN5 gene includethose described in PCT Publication No. WO 09/129319, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes. Non-limiting examples of siRNA molecules targeting the COP1,CSN5, RBX1, HDAC2, CDK4, WEE1, FOXM1, and RAM2 genes include thosedescribed in U.S. Provisional Application No. 61/245,143, filed Sep. 23,2009, the disclosure of which is herein incorporated by reference in itsentirety for all purposes.

Additional examples of gene sequences associated with tumorigenesis orcell transformation include translocation sequences such as MLL fusiongenes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al.,Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2,AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003));overexpressed sequences such as multidrug resistance genes (Nieth etal., FEBS Lett., 545:144 (2003); Wu et al, Cancer Res. 63:1515 (2003)),cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al., Genes Dev.,16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291(2003)), telomerase genes (Kosciolek et al., Mol Cancer Ther., 2:209(2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR/ErbB1(Genbank Accession Nos. NM 005228, NM_201282, NM 201283, and NM_201284;see also, Nagy et al. Exp. Cell Res., 285:39-49 (2003)), ErbB2/HER-2(Genbank Accession Nos. NM_004448 and NM_001005862), ErbB3 (GenbankAccession Nos. NM_001982 and NM_001005915), and ErbB4 (Genbank AccessionNos. NM_005235 and NM_001042599)), and mutated sequences such as RAS(Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002)). Non-limitingexamples of siRNA molecules targeting the EGFR gene include thosedescribed in U.S. Patent Application Publication No. 2009/0149403, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. siRNA molecules that target VEGFR genes are set forthin, e.g., GB 2396864; U.S. Patent Application Publication No.2004/0142895; and CA 2456444, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

Silencing of sequences that encode DNA repair enzymes find use incombination with the administration of chemotherapeutic agents (Colliset al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associatedwith tumor migration are also target sequences of interest, for example,integrins, selectins, and metalloproteinases. The foregoing examples arenot exclusive. Those of skill in the art will understand that any wholeor partial gene sequence that facilitates or promotes tumorigenesis orcell transformation, tumor growth, or tumor migration can be included asa template sequence.

Angiogenic genes are able to promote the formation of new vessels.Angiogenic genes of particular interest include, but are not limited to,vascular endothelial growth factor (VEGF) (Reich et al., Mol. Vis.,9:210 (2003)), placental growth factor (PGF), VEGFR-1 (Flt-1), VEGFR-2(KDR/Flk-1), and the like. siRNA molecules that target VEGFR genes areset forth in, e.g., GB 2396864; U.S. Patent Application Publication No.2004/0142895; and CA 2456444, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

Immunomodulator genes are genes that modulate one or more immuneresponses. Examples of immunomodulator genes include, withoutlimitation, growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF,PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12(Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18, IL-20, etc.),interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), and TNF. Fas and Fasligand genes are also immunomodulator target sequences of interest (Songet al., Nat. Med., 9:347 (2003)). Genes encoding secondary signalingmolecules in hematopoietic and lymphoid cells are also included in thepresent invention, for example, Tec family kinases such as Bruton'styrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002)).

Cell receptor ligand genes include ligands that are able to bind to cellsurface receptors (e.g., cytokine receptors, growth factor receptors,receptors with tyrosine kinase activity, G-protein coupled receptors,insulin receptor, EPO receptor, etc.) to modulate (e.g., inhibit) thephysiological pathway that the receptor is involved in (e.g., cellproliferation, tumorigenesis, cell transformation, mitogenesis, etc.).Non-limiting examples of cell receptor ligand genes include cytokines(e.g., TNF-α, interferons such as IFN-α, IFN-β, and IFN-γ, interleukinssuch as IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-17, IL-23, IL-27, chemokines, etc.), growthfactors (e.g., EGF, HB-EGF, VEGF, PEDF, SDGF, bFGF, HGF, TGF-α, TGF-β,BMP1-BMP15, PDGF, IGF, NGF, β-NGF, BDNF, NT3, NT4, GDF-9, CGF, G-CSF,GM-CSF, GDF-8, EPO, TPO, etc.), insulin, glucagon, G-protein coupledreceptor ligands, etc.

Templates coding for an expansion of trinucleotide repeats (e.g., CAGrepeats) find use in silencing pathogenic sequences in neurodegenerativedisorders caused by the expansion of trinucleotide repeats, such asspinobulbular muscular atrophy and Huntington's Disease (Caplen et al.,Hum. Mol. Genet., 11:175 (2002)).

In addition to its utility in silencing the expression of any of theabove-described genes for therapeutic purposes, the siRNA describedherein are also useful in research and development applications as wellas diagnostic, prophylactic, prognostic, clinical, and other healthcareapplications. As a non-limiting example, the siRNA can be used in targetvalidation studies directed at testing whether a gene of interest hasthe potential to be a therapeutic target. The siRNA can also be used intarget identification studies aimed at discovering genes as potentialtherapeutic targets.

e) Exemplary siRNA Embodiments

In some embodiments, each strand of the siRNA molecule comprises fromabout 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50,15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In one particularembodiment, the siRNA is chemically synthesized. The siRNA molecules ofthe invention are capable of silencing the expression of a targetsequence in vitro and/or in vivo.

In other embodiments, the siRNA comprises at least one modifiednucleotide. In certain embodiments, the siRNA comprises one, two, three,four, five, six, seven, eight, nine, ten, or more modified nucleotidesin the double-stranded region. In particular embodiments, less thanabout 50% (e.g., less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, or 5%) of the nucleotides in the double-stranded region of thesiRNA comprise modified nucleotides. In preferred embodiments, fromabout 1% to about 50% (e.g., from about 5%-50%, 10%-50%, 15%-50%,20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%, 45%-50%, 5%-45%, 10%-45%,15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%, 40%-45%, 5%-40%, 10%-40%,15%-40%, 20%-40%, 25%-40%, 30%-40%, 35%-40%, 5%-35%, 10%-35%, 15%-35%,20%-35%, 25%-35%, 30%-35%, 5%-30%, 10%-30%, 15%-30%, 20%-30%, 25%-30%,5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%,10%-15%, or 5%-10%) of the nucleotides in the double-stranded region ofthe siRNA comprise modified nucleotides.

In further embodiments, the siRNA comprises modified nucleotidesincluding, but not limited to, 2′-O-methyl (2′OMe) nucleotides,2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, and mixtures thereof. In preferred embodiments, the siRNAcomprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, e.g., 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosine nucleotides, ormixtures thereof. In one particular embodiment, the siRNA comprises atleast one 2′OMe-guanosine nucleotide, 2′OMe-uridine nucleotide, ormixtures thereof. In certain instances, the siRNA does not comprise2′OMe-cytosine nucleotides. In other embodiments, the siRNA comprises ahairpin loop structure.

In certain embodiments, the siRNA comprises modified nucleotides in onestrand (i.e., sense or antisense) or both strands of the double-strandedregion of the siRNA molecule. Preferably, uridine and/or guanosinenucleotides are modified at selective positions in the double-strandedregion of the siRNA duplex. With regard to uridine nucleotidemodifications, at least one, two, three, four, five, six, or more of theuridine nucleotides in the sense and/or antisense strand can be amodified uridine nucleotide such as a 2′OMe-uridine nucleotide. In someembodiments, every uridine nucleotide in the sense and/or antisensestrand is a 2′OMe-uridine nucleotide. With regard to guanosinenucleotide modifications, at least one, two, three, four, five, six, ormore of the guanosine nucleotides in the sense and/or antisense strandcan be a modified guanosine nucleotide such as a 2′OMe-guanosinenucleotide. In some embodiments, every guanosine nucleotide in the senseand/or antisense strand is a 2′OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six,seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified,e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/orby introducing modified nucleotides such as 2′OMe nucleotides. The5′-GU-3′ motif can be in the sense strand, the antisense strand, or bothstrands of the siRNA sequence. The 5′-GU-3′ motifs may be adjacent toeach other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more nucleotides.

In some embodiments, a modified siRNA molecule is less immunostimulatorythan a corresponding unmodified siRNA sequence. In such embodiments, themodified siRNA molecule with reduced immunostimulatory propertiesadvantageously retains RNAi activity against the target sequence. Inanother embodiment, the immunostimulatory properties of the modifiedsiRNA molecule and its ability to silence target gene expression can bebalanced or optimized by the introduction of minimal and selective 2′OMemodifications within the siRNA sequence such as, e.g., within thedouble-stranded region of the siRNA duplex. In certain instances, themodified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that the immunostimulatory properties of the modifiedsiRNA molecule and the corresponding unmodified siRNA molecule can bedetermined by, for example, measuring INF-α and/or IL-6 levels fromabout two to about twelve hours after systemic administration in amammal or transfection of a mammalian responder cell using anappropriate lipid-based delivery system (such as the LNP delivery systemdisclosed herein).

In other embodiments, a modified siRNA molecule has an IC₅₀ (i.e.,half-maximal inhibitory concentration) less than or equal to ten-foldthat of the corresponding unmodified siRNA (i.e., the modified siRNA hasan IC₅₀ that is less than or equal to ten-times the IC₅₀ of thecorresponding unmodified siRNA). In other embodiments, the modifiedsiRNA has an IC₅₀ less than or equal to three-fold that of thecorresponding unmodified siRNA sequence. In yet other embodiments, themodified siRNA has an IC₅₀ less than or equal to two-fold that of thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that a dose-response curve can be generated and theIC₅₀ values for the modified siRNA and the corresponding unmodifiedsiRNA can be readily determined using methods known to those of skill inthe art.

In another embodiment, an unmodified or modified siRNA molecule iscapable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% of the expression of the target sequencerelative to a negative control (e.g., buffer only, an siRNA sequencethat targets a different gene, a scrambled siRNA sequence, etc.).

In yet another embodiment, a modified siRNA molecule is capable ofsilencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of the expression of the target sequence relative tothe corresponding unmodified siRNA sequence.

In some embodiments, the siRNA molecule does not comprise phosphatebackbone modifications, e.g., in the sense and/or antisense strand ofthe double-stranded region. In other embodiments, the siRNA comprisesone, two, three, four, or more phosphate backbone modifications, e.g.,in the sense and/or antisense strand of the double-stranded region. Inpreferred embodiments, the siRNA does not comprise phosphate backbonemodifications.

In further embodiments, the siRNA does not comprise 2′-deoxynucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region. In yet further embodiments, the siRNA comprisesone, two, three, four, or more 2′-deoxy nucleotides, e.g., in the senseand/or antisense strand of the double-stranded region. In preferredembodiments, the siRNA does not comprise 2′-deoxy nucleotides.

In certain instances, the nucleotide at the 3′-end of thedouble-stranded region in the sense and/or antisense strand is not amodified nucleotide. In certain other instances, the nucleotides nearthe 3′-end (e.g., within one, two, three, or four nucleotides of the3′-end) of the double-stranded region in the sense and/or antisensestrand are not modified nucleotides.

The siRNA molecules described herein may have 3′ overhangs of one, two,three, four, or more nucleotides on one or both sides of thedouble-stranded region, or may lack overhangs (i.e., have blunt ends) onone or both sides of the double-stranded region. In certain embodiments,the 3′ overhang on the sense and/or antisense strand independentlycomprises one, two, three, four, or more modified nucleotides such as2′OMe nucleotides and/or any other modified nucleotide described hereinor known in the art.

In particular embodiments, siRNAs are administered using a carriersystem such as a nucleic acid-lipid particle. In a preferred embodiment,the nucleic acid-lipid particle comprises: (a) one or more siRNAmolecules; (b) a cationic lipid of Formula I or a salt thereof; and (c)a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol). Incertain instances, the nucleic acid-lipid particle may further comprisea conjugated lipid that prevents aggregation of particles (e.g., PEG-DAAand/or POZ-DAA).

2. Dicer-Substrate dsRNA

As used herein, the term “Dicer-substrate dsRNA” or “precursor RNAimolecule” is intended to include any precursor molecule that isprocessed in vivo by Dicer to produce an active siRNA which isincorporated into the RISC complex for RNA interference of a targetgene.

In one embodiment, the Dicer-substrate dsRNA has a length sufficientsuch that it is processed by Dicer to produce an siRNA. According tothis embodiment, the Dicer-substrate dsRNA comprises (i) a firstoligonucleotide sequence (also termed the sense strand) that is betweenabout 25 and about 60 nucleotides in length (e.g., about 25-60, 25-55,25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), preferablybetween about 25 and about 30 nucleotides in length (e.g., 25, 26, 27,28, 29, or 30 nucleotides in length), and (ii) a second oligonucleotidesequence (also termed the antisense strand) that anneals to the firstsequence under biological conditions, such as the conditions found inthe cytoplasm of a cell. The second oligonucleotide sequence may bebetween about 25 and about 60 nucleotides in length (e.g., about 25-60,25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), andis preferably between about 25 and about 30 nucleotides in length (e.g.,25, 26, 27, 28, 29, or 30 nucleotides in length). In addition, a regionof one of the sequences, particularly of the antisense strand, of theDicer-substrate dsRNA has a sequence length of at least about 19nucleotides, for example, from about 19 to about 60 nucleotides (e.g.,about 19-60, 19-55, 19-50, 19-45, 19-40, 19-35, 19-30, or 19-25nucleotides), preferably from about 19 to about 23 nucleotides (e.g.,19, 20, 21, 22, or 23 nucleotides) that are sufficiently complementaryto a nucleotide sequence of the RNA produced from the target gene totrigger an RNAi response.

In a second embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and has at least one of the following properties: (i)the dsRNA is asymmetric, e.g., has a 3′-overhang on the antisensestrand; and/or (ii) the dsRNA has a modified 3′-end on the sense strandto direct orientation of Dicer binding and processing of the dsRNA to anactive siRNA. According to this latter embodiment, the sense strandcomprises from about 22 to about 28 nucleotides and the antisense strandcomprises from about 24 to about 30 nucleotides.

In one embodiment, the Dicer-substrate dsRNA has an overhang on the3′-end of the antisense strand. In another embodiment, the sense strandis modified for Dicer binding and processing by suitable modifierslocated at the 3′-end of the sense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the Dicer-substrate dsRNA has an overhangon the 3′-end of the antisense strand and the sense strand is modifiedfor Dicer processing. In another embodiment, the 5′-end of the sensestrand has a phosphate. In another embodiment, the 5′-end of theantisense strand has a phosphate. In another embodiment, the antisensestrand or the sense strand or both strands have one or more 2′-O-methyl(2′OMe) modified nucleotides. In another embodiment, the antisensestrand contains 2′OMe modified nucleotides. In another embodiment, theantisense stand contains a 3′-overhang that is comprised of 2′OMemodified nucleotides. The antisense strand could also include additional2′OMe modified nucleotides. The sense and antisense strands anneal underbiological conditions, such as the conditions found in the cytoplasm ofa cell. In addition, a region of one of the sequences, particularly ofthe antisense strand, of the Dicer-substrate dsRNA has a sequence lengthof at least about 19 nucleotides, wherein these nucleotides are in the21-nucleotide region adjacent to the 3′-end of the antisense strand andare sufficiently complementary to a nucleotide sequence of the RNAproduced from the target gene. Further, in accordance with thisembodiment, the Dicer-substrate dsRNA may also have one or more of thefollowing additional properties: (a) the antisense strand has a rightshift from the typical 21-mer (i.e., the antisense strand includesnucleotides on the right side of the molecule when compared to thetypical 21-mer); (b) the strands may not be completely complementary,i.e., the strands may contain simple mismatch pairings; and (c) basemodifications such as locked nucleic acid(s) may be included in the5′-end of the sense strand.

In a third embodiment, the sense strand comprises from about 25 to about28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides), wherein the 2nucleotides on the 3′-end of the sense strand are deoxyribonucleotides.The sense strand contains a phosphate at the 5′-end. The antisensestrand comprises from about 26 to about 30 nucleotides (e.g., 26, 27,28, 29, or 30 nucleotides) and contains a 3′-overhang of 1-4nucleotides. The nucleotides comprising the 3′-overhang are modifiedwith 2′OMe modified ribonucleotides. The antisense strand containsalternating 2′OMe modified nucleotides beginning at the first monomer ofthe antisense strand adjacent to the 3′-overhang, and extending 15-19nucleotides from the first monomer adjacent to the 3′-overhang. Forexample, for a 27-nucleotide antisense strand and counting the firstbase at the 5′-end of the antisense strand as position number 1, 2′OMemodifications would be placed at bases 9, 11, 13, 15, 17, 19, 21, 23,25, 26, and 27. In one embodiment, the Dicer-substrate dsRNA has thefollowing structure:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′OMe RNA, “Y” is anoverhang domain comprised of 1, 2, 3, or 4 RNA monomers that areoptionally 2′OMe RNA monomers, and “D”=DNA. The top strand is the sensestrand, and the bottom strand is the antisense strand.

In a fourth embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and at least one of the following properties: (i) thedsRNA is asymmetric, e.g., has a 3′-overhang on the sense strand; and(ii) the dsRNA has a modified 3′-end on the antisense strand to directorientation of Dicer binding and processing of the dsRNA to an activesiRNA. According to this embodiment, the sense strand comprises fromabout 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30nucleotides) and the antisense strand comprises from about 22 to about28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides). In oneembodiment, the Dicer-substrate dsRNA has an overhang on the 3′-end ofthe sense strand. In another embodiment, the antisense strand ismodified for Dicer binding and processing by suitable modifiers locatedat the 3′-end of the antisense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the dsRNA has an overhang on the 3′-endof the sense strand and the antisense strand is modified for Dicerprocessing. In one embodiment, the antisense strand has a 5′-phosphate.The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′-end of antisense strand and aresufficiently complementary to a nucleotide sequence of the RNA producedfrom the target gene. Further, in accordance with this embodiment, theDicer-substrate dsRNA may also have one or more of the followingadditional properties: (a) the antisense strand has a left shift fromthe typical 21-mer (i.e., the antisense strand includes nucleotides onthe left side of the molecule when compared to the typical 21-mer); and(b) the strands may not be completely complementary, i.e., the strandsmay contain simple mismatch pairings.

In a preferred embodiment, the Dicer-substrate dsRNA has an asymmetricstructure, with the sense strand having a 25-base pair length, and theantisense strand having a 27-base pair length with a 2 base 3′-overhang.In certain instances, this dsRNA having an asymmetric structure furthercontains 2 deoxynucleotides at the 3′-end of the sense strand in placeof two of the ribonucleotides. In certain other instances, this dsRNAhaving an asymmetric structure further contains 2′OMe modifications atpositions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand(wherein the first base at the 5′-end of the antisense strand isposition 1). In certain additional instances, this dsRNA having anasymmetric structure further contains a 3′-overhang on the antisensestrand comprising 1, 2, 3, or 4 2′OMe nucleotides (e.g., a 3′-overhangof 2′OMe nucleotides at positions 26 and 27 on the antisense strand).

In another embodiment, Dicer-substrate dsRNAs may be designed by firstselecting an antisense strand siRNA sequence having a length of at least19 nucleotides. In some instances, the antisense siRNA is modified toinclude about 5 to about 11 ribonucleotides on the 5′-end to provide alength of about 24 to about 30 nucleotides. When the antisense strandhas a length of 21 nucleotides, 3-9, preferably 4-7, or more preferably6 nucleotides may be added on the 5′-end. Although the addedribonucleotides may be complementary to the target gene sequence, fullcomplementarity between the target sequence and the antisense siRNA isnot required. That is, the resultant antisense siRNA is sufficientlycomplementary with the target sequence. A sense strand is then producedthat has about 22 to about 28 nucleotides. The sense strand issubstantially complementary with the antisense strand to anneal to theantisense strand under biological conditions. In one embodiment, thesense strand is synthesized to contain a modified 3′-end to direct Dicerprocessing of the antisense strand. In another embodiment, the antisensestrand of the dsRNA has a 3′-overhang. In a further embodiment, thesense strand is synthesized to contain a modified 3′-end for Dicerbinding and processing and the antisense strand of the dsRNA has a3′-overhang.

In a related embodiment, the antisense siRNA may be modified to includeabout 1 to about 9 ribonucleotides on the 5′-end to provide a length ofabout 22 to about 28 nucleotides. When the antisense strand has a lengthof 21 nucleotides, 1-7, preferably 2-5, or more preferably 4ribonucleotides may be added on the 3′-end. The added ribonucleotidesmay have any sequence. Although the added ribonucleotides may becomplementary to the target gene sequence, full complementarity betweenthe target sequence and the antisense siRNA is not required. That is,the resultant antisense siRNA is sufficiently complementary with thetarget sequence. A sense strand is then produced that has about 24 toabout 30 nucleotides. The sense strand is substantially complementarywith the antisense strand to anneal to the antisense strand underbiological conditions. In one embodiment, the antisense strand issynthesized to contain a modified 3′-end to direct Dicer processing. Inanother embodiment, the sense strand of the dsRNA has a 3′-overhang. Ina further embodiment, the antisense strand is synthesized to contain amodified 3′-end for Dicer binding and processing and the sense strand ofthe dsRNA has a 3′-overhang.

Suitable Dicer-substrate dsRNA sequences can be identified, synthesized,and modified using any means known in the art for designing,synthesizing, and modifying siRNA sequences. In particular embodiments,Dicer-substrate dsRNAs are administered using a carrier system such as anucleic acid-lipid particle. In a preferred embodiment, the nucleicacid-lipid particle comprises: (a) one or more Dicer-substrate dsRNAmolecules; (b) a cationic lipid of Formula I or a salt thereof; and (c)a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol). Incertain instances, the nucleic acid-lipid particle may further comprisea conjugated lipid that prevents aggregation of particles (e.g., PEG-DAAand/or POZ-DAA).

Additional embodiments related to the Dicer-substrate dsRNAs of theinvention, as well as methods of designing and synthesizing such dsRNAs,are described in U.S. Patent Application Publication Nos. 2005/0244858,2005/0277610, and 2007/0265220, and U.S. Provisional Application No.61/184,652, filed Jun. 5, 2009, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

3. shRNA

A “small hairpin RNA” or “short hairpin RNA” or “shRNA” includes a shortRNA sequence that makes a tight hairpin turn that can be used to silencegene expression via RNA interference. The shRNAs of the invention may bechemically synthesized or transcribed from a transcriptional cassette ina DNA plasmid. The shRNA hairpin structure is cleaved by the cellularmachinery into siRNA, which is then bound to the RNA-induced silencingcomplex (RISC).

The shRNAs of the invention are typically about 15-60, 15-50, or 15-40(duplex) nucleotides in length, more typically about 15-30, 15-25, or19-25 (duplex) nucleotides in length, and are preferably about 20-24,21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementarysequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30,15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or21-23 nucleotides in length, and the double-stranded shRNA is about15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length,preferably about 18-22, 19-20, or 19-21 base pairs in length). shRNAduplexes may comprise 3′ overhangs of about 1 to about 4 nucleotides orabout 2 to about 3 nucleotides on the antisense strand and/or5′-phosphate termini on the sense strand. In some embodiments, the shRNAcomprises a sense strand and/or antisense strand sequence of from about15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50,15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), preferablyfrom about 19 to about 40 nucleotides in length (e.g., about 19-40,19-35, 19-30, or 19-25 nucleotides in length), more preferably fromabout 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23nucleotides in length).

Non-limiting examples of shRNA include a double-stranded polynucleotidemolecule assembled from a single-stranded molecule, where the sense andantisense regions are linked by a nucleic acid-based or non-nucleicacid-based linker; and a double-stranded polynucleotide molecule with ahairpin secondary structure having self-complementary sense andantisense regions. In preferred embodiments, the sense and antisensestrands of the shRNA are linked by a loop structure comprising fromabout 1 to about 25 nucleotides, from about 2 to about 20 nucleotides,from about 4 to about 15 nucleotides, from about 5 to about 12nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

Suitable shRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. In particular embodiments, shRNAs areadministered using a carrier system such as a nucleic acid-lipidparticle. In a preferred embodiment, the nucleic acid-lipid particlecomprises: (a) one or more shRNA molecules; (b) a cationic lipid ofFormula I or a salt thereof; and (c) a non-cationic lipid (e.g., DPPC,DSPC, DSPE, and/or cholesterol). In certain instances, the nucleicacid-lipid particle may further comprise a conjugated lipid thatprevents aggregation of particles (e.g., PEG-DAA and/or POZ-DAA).

Additional embodiments related to the shRNAs of the invention, as wellas methods of designing and synthesizing such shRNAs, are described inU.S. Provisional Application No. 61/184,652, filed Jun. 5, 2009, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

4. aiRNA

Like siRNA, asymmetrical interfering RNA (aiRNA) can recruit theRNA-induced silencing complex (RISC) and lead to effective silencing ofa variety of genes in mammalian cells by mediating sequence-specificcleavage of the target sequence between nucleotide 10 and 11 relative tothe 5′ end of the antisense strand (Sun et al., Nat. Biotech.,26:1379-1382 (2008)). Typically, an aiRNA molecule comprises a short RNAduplex having a sense strand and an antisense strand, wherein the duplexcontains overhangs at the 3′ and 5′ ends of the antisense strand. TheaiRNA is generally asymmetric because the sense strand is shorter onboth ends when compared to the complementary antisense strand. In someaspects, aiRNA molecules may be designed, synthesized, and annealedunder conditions similar to those used for siRNA molecules. As anon-limiting example, aiRNA sequences may be selected and generatedusing the methods described above for selecting siRNA sequences.

In another embodiment, aiRNA duplexes of various lengths (e.g., about10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs) may be designed withoverhangs at the 3′ and 5′ ends of the antisense strand to target anmRNA of interest. In certain instances, the sense strand of the aiRNAmolecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or20 nucleotides in length. In certain other instances, the antisensestrand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and is preferably about 20-24, 21-22, or 21-23 nucleotides inlength.

In some embodiments, the 5′ antisense overhang contains one, two, three,four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”, etc.).In other embodiments, the 3′ antisense overhang contains one, two,three, four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”,etc.). In certain aspects, the aiRNA molecules described herein maycomprise one or more modified nucleotides, e.g., in the double-stranded(duplex) region and/or in the antisense overhangs. As a non-limitingexample, aiRNA sequences may comprise one or more of the modifiednucleotides described above for siRNA sequences. In a preferredembodiment, the aiRNA molecule comprises 2′OMe nucleotides such as, forexample, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, ormixtures thereof.

In certain embodiments, aiRNA molecules may comprise an antisense strandwhich corresponds to the antisense strand of an siRNA molecule, e.g.,one of the siRNA molecules described herein. In particular embodiments,aiRNAs are administered using a carrier system such as a nucleicacid-lipid particle. In a preferred embodiment, the nucleic acid-lipidparticle comprises: (a) one or more aiRNA molecules; (b) a cationiclipid of Formula I or a salt thereof; and (c) a non-cationic lipid(e.g., DPPC, DSPC, DSPE, and/or cholesterol). In certain instances, thenucleic acid-lipid particle may further comprise a conjugated lipid thatprevents aggregation of particles (e.g., PEG-DAA and/or POZ-DAA).

Suitable aiRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. Additional embodiments related to the aiRNAmolecules of the invention are described in U.S. Patent ApplicationPublication No. 2009/0291131 and PCT Publication No. WO 09/127060, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

5. miRNA

Generally, microRNAs (miRNA) are single-stranded RNA molecules of about21-23 nucleotides in length which regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed, but miRNAs are nottranslated into protein (non-coding RNA); instead, each primarytranscript (a pri-miRNA) is processed into a short stem-loop structurecalled a pre-miRNA and finally into a functional mature miRNA. MaturemiRNA molecules are either partially or completely complementary to oneor more messenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. The identification of miRNA molecules isdescribed, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau etal., Science, 294:858-862; and Lee et al., Science, 294:862-864.

The genes encoding miRNA are much longer than the processed mature miRNAmolecule. miRNA are first transcribed as primary transcripts orpri-miRNA with a cap and poly-A tail and processed to short,˜70-nucleotide stem-loop structures known as pre-miRNA in the cellnucleus. This processing is performed in animals by a protein complexknown as the Microprocessor complex, consisting of the nuclease Droshaand the double-stranded RNA binding protein Pasha (Denli et al., Nature,432:231-235 (2004)). These pre-miRNA are then processed to mature miRNAin the cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC)(Bernstein et al., Nature, 409:363-366 (2001). Either the sense strandor antisense strand of DNA can function as templates to give rise tomiRNA.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end (Preall et al., Curr. Biol.,16:530-535 (2006)). The remaining strand, known as the anti-guide orpassenger strand, is degraded as a RISC complex substrate (Gregory etal., Cell, 123:631-640 (2005)). After integration into the active RISCcomplex, miRNAs base pair with their complementary mRNA molecules andinduce target mRNA degradation and/or translational silencing.

Mammalian miRNA molecules are usually complementary to a site in the 3′UTR of the target mRNA sequence. In certain instances, the annealing ofthe miRNA to the target mRNA inhibits protein translation by blockingthe protein translation machinery. In certain other instances, theannealing of the miRNA to the target mRNA facilitates the cleavage anddegradation of the target mRNA through a process similar to RNAinterference (RNAi). miRNA may also target methylation of genomic siteswhich correspond to targeted mRNA. Generally, miRNA function inassociation with a complement of proteins collectively termed the miRNP.

In certain aspects, the miRNA molecules described herein are about15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and are preferably about 20-24, 21-22, or 21-23 nucleotides inlength. In certain other aspects, miRNA molecules may comprise one ormore modified nucleotides. As a non-limiting example, miRNA sequencesmay comprise one or more of the modified nucleotides described above forsiRNA sequences. In a preferred embodiment, the miRNA molecule comprises2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, or mixtures thereof.

In particular embodiments, miRNAs are administered using a carriersystem such as a nucleic acid-lipid particle. In a preferred embodiment,the nucleic acid-lipid particle comprises: (a) one or more miRNAmolecules; (b) a cationic lipid of Formula I or a salt thereof; and (c)a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol). Incertain instances, the nucleic acid-lipid particle may further comprisea conjugated lipid that prevents aggregation of particles (e.g., PEG-DAAand/or POZ-DAA).

In other embodiments, one or more agents that block the activity of anmiRNA targeting an mRNA of interest are administered using a lipidparticle of the invention (e.g., a nucleic acid-lipid particle such asLNP). Examples of blocking agents include, but are not limited to,steric blocking oligonucleotides, locked nucleic acid oligonucleotides,and Morpholino oligonucleotides. Such blocking agents may bind directlyto the miRNA or to the miRNA binding site on the target mRNA.

Additional embodiments related to the miRNA molecules of the inventionare described in U.S. Patent Application Publication No. 2009/0291131and PCT Publication No. WO 09/127060, the disclosures of which areherein incorporated by reference in their entirety for all purposes.

6. Antisense Oligonucleotides

In one embodiment, the nucleic acid is an antisense oligonucleotidedirected to a target gene or sequence of interest. The terms “antisenseoligonucleotide” or “antisense” include oligonucleotides that arecomplementary to a targeted polynucleotide sequence. Antisenseoligonucleotides are single strands of DNA or RNA that are complementaryto a chosen sequence. Antisense RNA oligonucleotides prevent thetranslation of complementary RNA strands by binding to the RNA.Antisense DNA oligonucleotides can be used to target a specific,complementary (coding or non-coding) RNA. If binding occurs, thisDNA/RNA hybrid can be degraded by the enzyme RNase H. In a particularembodiment, antisense oligonucleotides comprise from about 10 to about60 nucleotides, more preferably from about 15 to about 30 nucleotides.The term also encompasses antisense oligonucleotides that may not beexactly complementary to the desired target gene. Thus, the inventioncan be utilized in instances where non-target specific-activities arefound with antisense, or where an antisense sequence containing one ormore mismatches with the target sequence is the most preferred for aparticular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(see, U.S. Pat. Nos. 5,739,119 and 5,759,829). Furthermore, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDR1), ICAM-1, E-selectin,STK-1, striatal GABAA receptor, and human EGF (see, Jaskulski et al.,Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer Commun.,1:225-32 (1989); Penis et al., Brain Res Mol Brain Res., 15; 57:310-20(1998); and U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and5,610,288). Moreover, antisense constructs have also been described thatinhibit and can be used to treat a variety of abnormal cellularproliferations, e.g., cancer (see, U.S. Pat. Nos. 5,747,470; 5,591,317;and 5,783,683). The disclosures of these references are hereinincorporated by reference in their entirety for all purposes.

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

7. Ribozymes

According to another embodiment of the invention, nucleic acid-lipidparticles are associated with ribozymes. Ribozymes are RNA-proteincomplexes having specific catalytic domains that possess endonucleaseactivity (see, Kim et al., Proc. Natl. Acad. Sci. USA., 84:8788-92(1987); and Forster et al., Cell, 49:211-20 (1987)). For example, alarge number of ribozymes accelerate phosphoester transfer reactionswith a high degree of specificity, often cleaving only one of severalphosphoesters in an oligonucleotide substrate (see, Cech et al., Cell,27:487-96 (1981); Michel et al., J. Mol. Biol., 216:585-610 (1990);Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAmolecules are known presently. Each can catalyze the hydrolysis of RNAphosphodiester bonds in trans (and thus can cleave other RNA molecules)under physiological conditions. In general, enzymatic nucleic acids actby first binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, hepatitis δ virus, group I intron or RNaseP RNA (in associationwith an RNA guide sequence), or Neurospora VS RNA motif, for example.Specific examples of hammerhead motifs are described in, e.g., Rossi etal., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifsare described in, e.g., EP 0360257, Hampel et al., Biochemistry,28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990);and U.S. Pat. No. 5,631,359. An example of the hepatitis δ virus motifis described in, e.g., Perrotta et al., Biochemistry, 31:11843-52(1992). An example of the RNaseP motif is described in, e.g.,Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of theNeurospora VS RNA ribozyme motif is described in, e.g., Saville et al.,Cell, 61:685-96 (1990); Saville et al., Proc. Natl. Acad. Sci. USA,88:8826-30 (1991); Collins et al., Biochemistry, 32:2795-9 (1993). Anexample of the Group I intron is described in, e.g., U.S. Pat. No.4,987,071. Important characteristics of enzymatic nucleic acid moleculesused according to the invention are that they have a specific substratebinding site which is complementary to one or more of the target geneDNA or RNA regions, and that they have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule. Thus, the ribozyme constructs need not belimited to specific motifs mentioned herein. The disclosures of thesereferences are herein incorporated by reference in their entirety forall purposes.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in, e.g.,PCT Publication Nos. WO 93/23569 and WO 94/02595, and synthesized to betested in vitro and/or in vivo as described therein. The disclosures ofthese PCT publications are herein incorporated by reference in theirentirety for all purposes.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases(see, e.g., PCT Publication Nos. WO 92/07065, WO 93/15187, WO 91/03162,and WO 94/13688; EP 92110298.4; and U.S. Pat. No. 5,334,711, whichdescribe various chemical modifications that can be made to the sugarmoieties of enzymatic RNA molecules, the disclosures of which are eachherein incorporated by reference in their entirety for all purposes),modifications which enhance their efficacy in cells, and removal of stemII bases to shorten RNA synthesis times and reduce chemicalrequirements.

8. Immunostimulatory Oligonucleotides

Nucleic acids associated with the lipid particles of the presentinvention may be immunostimulatory, including immunostimulatoryoligonucleotides (ISS; single- or double-stranded) capable of inducingan immune response when administered to a subject, which may be a mammalsuch as a human. ISS include, e.g., certain palindromes leading tohairpin secondary structures (see, Yamamoto et al., J. Immunol,148:4072-6 (1992)), or CpG motifs, as well as other known ISS features(such as multi-G domains; see; PCT Publication No. WO 96/11266, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes).

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target sequence in order to provoke an immuneresponse. Thus, certain immunostimulatory nucleic acids may comprise asequence corresponding to a region of a naturally-occurring gene ormRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein the CpG dinucleotide is methylated. In an alternative embodiment, thenucleic acid comprises at least two CpG dinucleotides, wherein at leastone cytosine in the CpG dinucleotides is methylated. In a furtherembodiment, each cytosine in the CpG dinucleotides present in thesequence is methylated. In another embodiment, the nucleic acidcomprises a plurality of CpG dinucleotides, wherein at least one of theCpG dinucleotides comprises a methylated cytosine. Examples ofimmunostimulatory oligonucleotides suitable for use in the compositionsand methods of the present invention are described in PCT PublicationNos. WO 02/069369, WO 01/15726, and WO 09/086558; U.S. Pat. No.6,406,705; and Raney et al., Pharm. Exper. Ther., 298:1185-92 (2001),the disclosures of which are herein incorporated by reference in theirentirety for all purposes. In certain embodiments, the oligonucleotidesused in the compositions and methods of the invention have aphosphodiester (“PO”) backbone or a phosphorothioate (“PS”) backbone,and/or at least one methylated cytosine residue in a CpG motif.

B. Other Active Agents

In certain embodiments, the active agent associated with the lipidparticles of the invention may comprise one or more therapeuticproteins, polypeptides, or small organic molecules or compounds.Non-limiting examples of such therapeutically effective agents or drugsinclude oncology drugs (e.g., chemotherapy drugs, hormonal therapaeuticagents, immunotherapeutic agents, radiotherapeutic agents, etc.),lipid-lowering agents, anti-viral drugs, anti-inflammatory compounds,antidepressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs such asanti-arrhythmic agents, hormones, vasoconstrictors, and steroids. Theseactive agents may be administered alone in the lipid particles of theinvention, or in combination (e.g., co-administered) with lipidparticles of the invention comprising nucleic acid such as interferingRNA.

Non-limiting examples of chemotherapy drugs include platinum-based drugs(e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin,satraplatin, etc.), alkylating agents (e.g., cyclophosphamide,ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine,uramustine, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g.,5-fluorouracil (5-FU), azathioprine, methotrexate, leucovorin,capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine,pemetrexed, raltitrexed, etc.), plant alkaloids (e.g., vincristine,vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel(taxol), docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan(CPT-11; Camptosar), topotecan, amsacrine, etoposide (VP16), etoposidephosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin,adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), tyrosine kinase inhibitors (e.g.,gefitinib (Iressa®), sunitinib (Sutent®; SU11248), erlotinib (Tarceva®;OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib(SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib(Gleevec®; STI571), dasatinib (BMS-354825), leflunomide (SU101),vandetanib (Zactima™; ZD6474), etc.), pharmaceutically acceptable saltsthereof, stereoisomers thereof, derivatives thereof, analogs thereof,and combinations thereof.

Examples of conventional hormonal therapaeutic agents include, withoutlimitation, steroids (e.g., dexamethasone), finasteride, aromataseinhibitors, tamoxifen, and goserelin as well as othergonadotropin-releasing hormone agonists (GnRH).

Examples of conventional immunotherapeutic agents include, but are notlimited to, immunostimulants (e.g., Bacillus Calmette-Guérin (BCG),levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.).

Examples of conventional radiotherapeutic agents include, but are notlimited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directed againsttumor antigens.

Additional oncology drugs that may be used according to the inventioninclude, but are not limited to, alkeran, allopurinol, altretamine,amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU,carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosinearabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane,FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin,interferon, letrozole, leustatin, leuprolide, litretinoin, megastrol,L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate,Pegademase, pentostatin, porfimer sodium, prednisone, rituxan,streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene,tretinoin, ATRA, valrubicin, and velban. Other examples of oncologydrugs that may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors, and camptothecins.

Non-limiting examples of lipid-lowering agents for treating a lipiddisease or disorder associated with elevated triglycerides, cholesterol,and/or glucose include statins, fibrates, ezetimibe, thiazolidinediones,niacin, beta-blockers, nitroglycerin, calcium antagonists, fish oil, andmixtures thereof.

Examples of anti-viral drugs include, but are not limited to, abacavir,aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol,atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine,didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide,entecavir, entry inhibitors, famciclovir, fixed dose combinations,fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors,ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir,inosine, integrase inhibitors, interferon type III (e.g., IFN-λmolecules such as IFN-λ1, IFN-λ2, and IFN-λ3), interferon type II (e.g.,IFN-γ), interferon type I (e.g., IFN-α such as PEGylated IFN-α, IFN-β,IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ), interferon, lamivudine,lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir,nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir,peramivir, pleconaril, podophyllotoxin, protease inhibitors, reversetranscriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir,stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil,tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,zanamivir, zidovudine, pharmaceutically acceptable salts thereof,stereoisomers thereof, derivatives thereof, analogs thereof, andmixtures thereof.

V. Lipid Particles

In certain aspects, the present invention provides lipid particlescomprising one or more of the cationic (amino) lipids or salts thereofdescribed herein. In some embodiments, the lipid particles of theinvention further comprise one or more non-cationic lipids. In otherembodiments, the lipid particles further comprise one or more conjugatedlipids capable of reducing or inhibiting particle aggregation. Inadditional embodiments, the lipid particles further comprise one or moreactive agents or therapeutic agents such as therapeutic nucleic acids(e.g., interfering RNA such as siRNA).

Lipid particles include, but are not limited to, lipid vesicles such asliposomes. As used herein, a lipid vesicle includes a structure havinglipid-containing membranes enclosing an aqueous interior. In particularembodiments, lipid vesicles comprising one or more of the cationiclipids described herein are used to encapsulate nucleic acids within thelipid vesicles. In other embodiments, lipid vesicles comprising one ormore of the cationic lipids described herein are complexed with nucleicacids to form lipoplexes.

The lipid particles of the invention typically comprise an active agentor therapeutic agent, a cationic lipid, a non-cationic lipid, and aconjugated lipid that inhibits aggregation of particles. In someembodiments, the active agent or therapeutic agent is fully encapsulatedwithin the lipid portion of the lipid particle such that the activeagent or therapeutic agent in the lipid particle is resistant in aqueoussolution to enzymatic degradation, e.g., by a nuclease or protease. Inother embodiments, the lipid particles described herein aresubstantially non-toxic to mammals such as humans. The lipid particlesof the invention typically have a mean diameter of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, or from about 70 to about 90 nm. The lipid particles ofthe invention also typically have a lipid:therapeutic agent (e.g.,lipid:nucleic acid) ratio (mass/mass ratio) of from about 1:1 to about100:1, from about 1:1 to about 50:1, from about 2:1 to about 25:1, fromabout 3:1 to about 20:1, from about 5:1 to about 15:1, or from about 5:1to about 10:1.

In preferred embodiments, the lipid particles of the invention areserum-stable nucleic acid-lipid particles (LNP) which comprise aninterfering RNA (e.g., siRNA, Dicer-substrate dsRNA, shRNA, aiRNA,and/or miRNA), a cationic lipid (e.g., one or more cationic lipids ofFormula I or salts thereof as set forth herein), a non-cationic lipid(e.g., mixtures of one or more phospholipids and cholesterol), and aconjugated lipid that inhibits aggregation of the particles (e.g., oneor more PEG-lipid and/or POZ-lipid conjugates). The LNP may comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodified and/or modifiedinterfering RNA molecules. Nucleic acid-lipid particles and their methodof preparation are described in, e.g., U.S. Pat. Nos. 5,753,613;5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and 6,320,017;and PCT Publication No. WO 96/40964, the disclosures of which are eachherein incorporated by reference in their entirety for all purposes.

In the nucleic acid-lipid particles of the invention, the nucleic acidmay be fully encapsulated within the lipid portion of the particle,thereby protecting the nucleic acid from nuclease degradation. Inpreferred embodiments, a LNP comprising a nucleic acid such as aninterfering RNA is fully encapsulated within the lipid portion of theparticle, thereby protecting the nucleic acid from nuclease degradation.In certain instances, the nucleic acid in the LNP is not substantiallydegraded after exposure of the particle to a nuclease at 37° C. for atleast about 20, 30, 45, or 60 minutes. In certain other instances, thenucleic acid in the LNP is not substantially degraded after incubationof the particle in serum at 37° C. for at least about 30, 45, or 60minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, thenucleic acid is complexed with the lipid portion of the particle. One ofthe benefits of the formulations of the present invention is that thenucleic acid-lipid particle compositions are substantially non-toxic tomammals such as humans.

The term “fully encapsulated” indicates that the nucleic acid in thenucleic acid-lipid particle is not significantly degraded after exposureto serum or a nuclease assay that would significantly degrade free DNAor RNA. In a fully encapsulated system, preferably less than about 25%of the nucleic acid in the particle is degraded in a treatment thatwould normally degrade 100% of free nucleic acid, more preferably lessthan about 10%, and most preferably less than about 5% of the nucleicacid in the particle is degraded. “Fully encapsulated” also indicatesthat the nucleic acid-lipid particles are serum-stable, that is, thatthey do not rapidly decompose into their component parts upon in vivoadministration.

In the context of nucleic acids, full encapsulation may be determined byperforming a membrane-impermeable fluorescent dye exclusion assay, whichuses a dye that has enhanced fluorescence when associated with nucleicacid. Specific dyes such as OliGreen® and RiboGreen® (Invitrogen Corp.;Carlsbad, Calif.) are available for the quantitative determination ofplasmid DNA, single-stranded deoxyribonucleotides, and/or single- ordouble-stranded ribonucleotides. Encapsulation is determined by addingthe dye to a liposomal formulation, measuring the resultingfluorescence, and comparing it to the fluorescence observed uponaddition of a small amount of nonionic detergent. Detergent-mediateddisruption of the liposomal bilayer releases the encapsulated nucleicacid, allowing it to interact with the membrane-impermeable dye. Nucleicacid encapsulation may be calculated as E=(I_(o)−I)/I_(o), where I andI_(o) refer to the fluorescence intensities before and after theaddition of detergent (see, Wheeler et al., Gene Ther., 6:271-281(1999)).

In other embodiments, the present invention provides a nucleicacid-lipid particle (e.g., LNP) composition comprising a plurality ofnucleic acid-lipid particles.

In some instances, the LNP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the particles have the nucleic acid encapsulated therein.

In other instances, the LNP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the input nucleic acid is encapsulated in the particles.

Depending on the intended use of the lipid particles of the invention,the proportions of the components can be varied and the deliveryefficiency of a particular formulation can be measured using, e.g., anendosomal release parameter (ERP) assay.

In particular embodiments, the present invention provides a lipidparticle (e.g., LNP) composition comprising a plurality of lipidparticles described herein and an antioxidant. In certain instances, theantioxidant in the lipid particle composition reduces, prevents, and/orinhibits the degradation of a cationic lipid present in the lipidparticle. In instances wherein the active agent is a therapeutic nucleicacid such as an interfering RNA (e.g., siRNA), the antioxidant in thelipid particle composition reduces, prevents, and/or inhibits thedegradation of the nucleic acid payload, e.g., by reducing, preventing,and/or inhibiting the formation of adducts between the nucleic acid andthe cationic lipid. Non-limiting examples of antioxidants includehydrophilic antioxidants such as chelating agents (e.g., metal chelatorssuch as ethylenediaminetetraacetic acid (EDTA), citrate, and the like),lipophilic antioxidants (e.g., vitamin E isomers, polyphenols, and thelike), salts thereof; and mixtures thereof. If needed, the antioxidantis typically present in an amount sufficient to prevent, inhibit, and/orreduce the degradation of the cationic lipid and/or active agent presentin the particle, e.g., at least about 20 mM EDTA or a salt thereof, orat least about 100 mM citrate or a salt thereof. An antioxidant such asEDTA and/or citrate may be included at any step or at multiple steps inthe lipid particle formation process described in Section VI (e.g.,prior to, during, and/or after lipid particle formation).

Additional embodiments related to methods of preventing the degradationof cationic lipids and/or active agents (e.g., therapeutic nucleicacids) present in lipid particles, compositions comprising lipidparticles stabilized by these methods, methods of making these lipidparticles, and methods of delivering and/or administering these lipidparticles are described in International Patent Application No.PCT/CA2010/001919, entitled “SNALP Formulations ContainingAntioxidants,” filed Dec. 1, 2010, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

A. Cationic Lipids

Any of the novel cationic lipids of Formula I or salts thereof as setforth herein may be used in the lipid particles of the present invention(e.g., LNP), either alone or in combination with one or more othercationic lipid species or non-cationic lipid species.

Other cationic lipids or salts thereof which may also be included in thelipid particles of the present invention include, but are not limitedto, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA),1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDMA),1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDAP),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;also known as “XTC2” or “C2K”),2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA;“C3K”), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA; “C4K”),2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA),2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DO-K-DMA),2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DS-K-DMA),2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA),2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride(DLin-K-TMA.Cl),2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane(DLin-K²-DMA), 2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane(D-Lin-K-N-methylpiperzine), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate(DLin-M-C3-DMA; “MC3”), dilinoleylmethyl-3-dimethylaminopropionate(DLin-M-C2-DMA; also known as DLin-M-K-DMA or DLin-M-DMA),1,2-dioeylcarbamoyloxy-3-dimethylaminopropane (DO-C-DAP),1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP),1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.Cl),1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS), analogs thereof, andmixtures thereof.

Additional cationic lipids or salts thereof which may be present in thelipid particles described herein include novel cationic lipids such asCP-LenMC3, CP-γ-LenMC3, CP-MC3, CP-DLen-C2K-DMA, CP-γDLen-C2K-DMA,CP-C2K-DMA, CP-DODMA, CP-DPetroDMA, CP-DLinDMA, CP-DLenDMA, CP-yDLenDMA,analogs thereof, and combinations thereof. Additional cationic lipids orsalts thereof which may be present in the lipid particles describedherein include MC3 analogs such as LenMC3, γ-LenMC3, MC3MC, MC2C, MC2MC,MC3 Thioester, MC3 Ether, MC4 Ether, MC3 Alkyne, MC3 Amide, Pan-MC3,Pan-MC4, Pan-MC5, and combinations thereof. Additional cationic lipidsor salts thereof which may be present in the lipid particles describedherein include the novel cationic lipids described in InternationalPatent Application No. PCT/CA2010/001029, entitled “Improved CationicLipids and Methods for the Delivery of Nucleic Acids,” filed Jun. 30,2010. Additional cationic lipids or salts thereof which may be presentin the lipid particles described herein include the cationic lipidsdescribed in U.S. Patent Application Publication No. 2009/0023673. Thedisclosures of each of these patent documents are herein incorporated byreference in their entirety for all purposes.

In some embodiments, the additional cationic lipid forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the additional cationic lipid is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLinDMA and DLenDMA, as well asadditional cationic lipids, is described in U.S. Patent ApplicationPublication No. 2006/0083780, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The synthesis of cationic lipids such as γ-DLenDMA, C2-DLinDMA andC2-DLinDAP, as well as additional cationic lipids, is described inInternational Patent Application No. PCT/CA2010/001029, entitled“Improved Cationic Lipids and Methods for the Delivery of NucleicAcids,” filed Jun. 30, 2010, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The synthesis of cationic lipids such as DLin-K-DMA, as well asadditional cationic lipids, is described in PCT Publication No. WO09/086558, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of cationic lipids such as DLin-K-C2-DMA, DLin-K-C3-DMA,DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ, DO-K-DMA, DS-K-DMA, DLin-K-MA,DLin-K-TMA.Cl, DLin-K²-DMA, D-Lin-K-N-methylpiperzine, DLin-M-C2-DMA,DO-C-DAP, DMDAP, and DOTAP.Cl, as well as additional cationic lipids, isdescribed in PCT Publication No. WO 2010/042877, entitled “ImprovedAmino Lipids and Methods for the Delivery of Nucleic Acids,” filed Oct.9, 2009, the disclosure of which is incorporated herein by reference inits entirety for all purposes.

The synthesis of DLin-M-C3-DMA, as well as additional cationic lipids,is described, for example, in U.S. Provisional Application No.61/384,050, filed Sep. 17, 2010, entitled “Novel Cationic Lipids andMethods of Use Thereof,” the disclosure of which is herein incorporatedby reference in its entirety for all purposes.

The synthesis of cationic lipids such as DLin-C-DAP, DLinDAC, DLinMA,DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl, DLinTAP.Cl, DLinMPZ,DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, isdescribed in PCT Publication No. WO 09/086558, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

The synthesis of cationic lipids such as CLinDMA, as well as additionalcationic lipids, is described in U.S. Patent Application Publication No.2006/0240554, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

The synthesis of a number of other cationic lipids and related analogshas been described in U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833;5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO96/10390, the disclosures of which are each herein incorporated byreference in their entirety for all purposes. Additionally, a number ofcommercial preparations of cationic lipids can be used, such as, e.g.,LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO/BRL);LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO/BRL); andTRANSFECTAM® (including DOGS, available from Promega Corp.).

In some embodiments, the cationic lipid comprises from about 50 mol % toabout 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol% to about 80 mol %, from about 50 mol % to about 75 mol %, from about50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, fromabout 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %,or from about 55 mol % to about 70 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle. In particularembodiments, the cationic lipid comprises about 50 mol %, 51 mol %, 52mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59mol %, 60 mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (orany fraction thereof) of the total lipid present in the particle.

In other embodiments, the cationic lipid comprises from about 2 mol % toabout 60 mol %, from about 5 mol % to about 50 mol %, from about 10 mol% to about 50 mol %, from about 20 mol % to about 50 mol %, from about20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %, orabout 40 mol % (or any fraction thereof or range therein) of the totallipid present in the particle.

Additional percentages and ranges of cationic lipids suitable for use inthe lipid particles of the present invention are described, for example,in PCT Publication No. WO 09/127060, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

It should be understood that the percentage of cationic lipid present inthe lipid particles of the invention is a target amount, and that theactual amount of cationic lipid present in the formulation may vary, forexample, by ±5 mol %. For example, in the 1:57 lipid particle (e.g.,LNP) formulation, the target amount of cationic lipid is 57.1 mol %, butthe actual amount of cationic lipid may be ±5 mol %, ±4 mol %, ±3 mol %,±2 mol %, 1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol %of that target amount, with the balance of the formulation being made upof other lipid components (adding up to 100 mol % of total lipidspresent in the particle).

B. Non-Cationic Lipids

The non-cationic lipids used in the lipid particles of the invention(e.g., LNP) can be any of a variety of neutral uncharged, zwitterionic,or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidyl glycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof. Non-limiting examples ofcholesterol derivatives include polar analogues such as 5α-cholestanol,5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether,cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polaranalogues such as 5α-cholestane, cholestenone, 5α-cholestanone,5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. Inpreferred embodiments, the cholesterol derivative is a polar analoguesuch as cholesteryl-(4′-hydroxy)-butyl ether. The synthesis ofcholesteryl-(2′-hydroxy)-ethyl ether is described in PCT Publication No.WO 09/127060, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

In some embodiments, the non-cationic lipid present in the lipidparticles (e.g., LNP) comprises or consists of a mixture of one or morephospholipids and cholesterol or a derivative thereof. In otherembodiments, the non-cationic lipid present in the lipid particles(e.g., LNP) comprises or consists of one or more phospholipids, e.g., acholesterol-free lipid particle formulation. In yet other embodiments,the non-cationic lipid present in the lipid particles (e.g., LNP)comprises or consists of cholesterol or a derivative thereof, e.g., aphospholipid-free lipid particle formulation.

Other examples of non-cationic lipids suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

In some embodiments, the non-cationic lipid comprises from about 10 mol% to about 60 mol %, from about 20 mol % to about 55 mol %, from about20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %,from about 30 mol % to about 50 mol %, from about 30 mol % to about 45mol %, from about 30 mol % to about 40 mol %, from about 35 mol % toabout 45 mol %, from about 37 mol % to about 42 mol %, or about 35 mol%, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %,43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60mol % of the total lipid present in the particle.

In some embodiments, the phospholipid component in the mixture maycomprise from about 2 mol % to about 20 mol %, from about 2 mol % toabout 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol %to about 15 mol %, or from about 4 mol % to about 10 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the phospholipid componentin the mixture comprises from about 5 mol % to about 10 mol %, fromabout 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %,from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol%, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a 1:57 lipid particle formulationcomprising a mixture of phospholipid and cholesterol may comprise aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof), e.g., in a mixture with cholesterol or a cholesterolderivative at about 34 mol % (or any fraction thereof) of the totallipid present in the particle. As another non-limiting example, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise a phospholipid such as DPPC or DSPC at about 7mol % (or any fraction thereof), e.g., in a mixture with cholesterol ora cholesterol derivative at about 32 mol % (or any fraction thereof) ofthe total lipid present in the particle.

In other embodiments, the cholesterol component in the mixture maycomprise from about 25 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol% to about 40 mol %, from about 27 mol % to about 37 mol %, from about25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the cholesterol component inthe mixture comprises from about 25 mol % to about 35 mol %, from about27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, fromabout 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %,from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle. Typically, a 1:57lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise cholesterol or a cholesterol derivative atabout 34 mol (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle. Typically, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise cholesterol or a cholesterol derivative atabout 32 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle.

In embodiments where the lipid particles are phospholipid-free, thecholesterol or derivative thereof may comprise up to about 25 mol %, 30mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % ofthe total lipid present in the particle.

In some embodiments, the cholesterol or derivative thereof in thephospholipid-free lipid particle formulation may comprise from about 25mol % to about 45 mol %, from about 25 mol % to about 40 mol %, fromabout 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %,from about 31 mol % to about 39 mol %, from about 32 mol % to about 38mol %, from about 33 mol % to about 37 mol %, from about 35 mol % toabout 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol% to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a 1:62 lipid particle formulationmay comprise cholesterol at about 37 mol % (or any fraction thereof) ofthe total lipid present in the particle. As another non-limitingexample, a 7:58 lipid particle formulation may comprise cholesterol atabout 35 mol % (or any fraction thereof) of the total lipid present inthe particle.

In other embodiments, the non-cationic lipid comprises from about 5 mol% to about 90 mol %, from about 10 mol % to about 85 mol %, from about20 mol % to about 80 mol %, about 10 mol % (e.g., phospholipid only), orabout 60 mol % (e.g., phospholipid and cholesterol or derivativethereof) (or any fraction thereof or range therein) of the total lipidpresent in the particle.

Additional percentages and ranges of non-cationic lipids suitable foruse in the lipid particles of the present invention are described, forexample, in PCT Publication No. WO 09/127060, the disclosure of which isherein incorporated by reference in its entirety for all purposes.

It should be understood that the percentage of non-cationic lipidpresent in the lipid particles of the invention is a target amount, andthat the actual amount of non-cationic lipid present in the formulationmay vary, for example, by ±5 mol %. For example, in the 1:57 lipidparticle (e.g., LNP) formulation, the target amount of phospholipid is7.1 mol % and the target amount of cholesterol is 34.3 mol %, but theactual amount of phospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that targetamount, and the actual amount of cholesterol may be ±3 mol %, ±2 mol %,±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle). Similarly, in the 7:54 lipid particle (e.g., LNP)formulation, the target amount of phospholipid is 6.75 mol % and thetarget amount of cholesterol is 32.43 mol %, but the actual amount ofphospholipid may be ±2 mol %, ±1.5 mol %, 1 mol %, ±0.75 mol %, ±0.5 mol%, f 0.25 mol %, or ±0.1 mol % of that target amount, and the actualamount of cholesterol may be ±3 mol %, ±2 mol %, ±1 mol %, 0.75 mol %, f0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, with thebalance of the formulation being made up of other lipid components(adding up to 100 mol % of total lipids present in the particle).

C. Lipid Conjugates

In addition to cationic and non-cationic lipids, the lipid particles ofthe invention (e.g., LNP) may further comprise a lipid conjugate. Theconjugated lipid is useful in that it prevents the aggregation ofparticles. Suitable conjugated lipids include, but are not limited to,PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates,cationic-polymer-lipid conjugates (CPLs), and mixtures thereof. Incertain embodiments, the particles comprise either a PEG-lipid conjugateor an ATTA-lipid conjugate together with a CPL.

In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examplesof PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Application Publication Nos. 2003/0077829 and2005/008689, PEG coupled to phospholipids such asphosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides asdescribed in, e.g., U.S. Pat. No. 5,885,613, PEG conjugated tocholesterol or a derivative thereof, and mixtures thereof. Thedisclosures of these patent documents are herein incorporated byreference in their entirety for all purposes.

Additional PEG-lipids suitable for use in the invention include, withoutlimitation, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).The synthesis of PEG-C-DOMG is described in PCT Publication No. WO09/086558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. Yet additional suitable PEG-lipidconjugates include, without limitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S.Pat. No. 7,404,969, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, but are not limited to, thefollowing: monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES),monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as wellas such compounds containing a terminal hydroxyl group instead of aterminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH₂, etc.).Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing thePEG-lipid conjugates of the present invention. The disclosures of thesepatents are herein incorporated by reference in their entirety for allpurposes. In addition, monomethoxypolyethyleneglycol-acetic acid(MePEG-CH₂COOH) is particularly useful for preparing PEG-lipidconjugates including, e.g., PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.Phosphatidyl-ethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

The term “ATTA” or “polyamide” includes, without limitation, compoundsdescribed in U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. These compounds include a compound having the formula:

wherein R is a member selected from the group consisting of hydrogen,alkyl and acyl; R¹ is a member selected from the group consisting ofhydrogen and alkyl; or optionally, R and R¹ and the nitrogen to whichthey are bound form an azido moiety; R² is a member of the groupselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl and a side chain of an amino acid; R³ is a memberselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR⁴R⁵, wherein R⁴ and R⁵ areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be used in the compounds of the present invention.

The term “diacylglycerol” or “DAG” includes a compound having 2 fattyacyl chains, R¹ and R², both of which have independently between 2 and30 carbons bonded to the 1- and 2-position of glycerol by esterlinkages. The acyl groups can be saturated or have varying degrees ofunsaturation. Suitable acyl groups include, but are not limited tolauroyl (C₁₂), myristoyl (C₁₄), palmitoyl (C₁₆), stearoyl (C₁₈), andicosoyl (C₂₀). In preferred embodiments, R¹ and R² are the same, i.e.,R¹ and R² are both myristoyl (i.e., dimyristoyl), R¹ and R² are bothstearoyl (i.e., distearoyl), etc. Diacylglycerols have the followinggeneral formula:

The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkylchains, R¹ and R², both of which have independently between 2 and 30carbons. The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate havingthe following formula:

wherein R¹ and R² are independently selected and are long-chain alkylgroups having from about 10 to about 22 carbon atoms; PEG is apolyethyleneglycol; and L is a non-ester containing linker moiety or anester containing linker moiety as described above. The long-chain alkylgroups can be saturated or unsaturated. Suitable alkyl groups include,but are not limited to, decyl (C₁₀), lauryl (C₁₂), myristyl (C₁₄),palmityl (C₁₆), stearyl (C₁₈), and icosyl (C₂₀). In preferredembodiments, R¹ and R² are the same, i.e., R¹ and R² are both myristyl(i.e., dimyristyl), R¹ and R² are both stearyl (i.e., distearyl), etc.

In Formula V above, the PEG has an average molecular weight ranging fromabout 550 daltons to about 10,000 daltons. In certain instances, the PEGhas an average molecular weight of from about 750 daltons to about 5,000daltons (e.g., from about 1,000 daltons to about 5,000 daltons, fromabout 1,500 daltons to about 3,000 daltons, from about 750 daltons toabout 3,000 daltons, from about 750 daltons to about 2,000 daltons,etc.). In preferred embodiments, the PEG has an average molecular weightof about 2,000 daltons or about 750 daltons. The PEG can be optionallysubstituted with alkyl, alkoxy, acyl, or aryl groups. In certainembodiments, the terminal hydroxyl group is substituted with a methoxyor methyl group.

In a preferred embodiment, “L” is a non-ester containing linker moiety.Suitable non-ester containing linkers include, but are not limited to,an amido linker moiety, an amino linker moiety, a carbonyl linkermoiety, a carbamate linker moiety, a urea linker moiety, an ether linkermoiety, a disulphide linker moiety, a succinamidyl linker moiety, andcombinations thereof. In a preferred embodiment, the non-estercontaining linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAAconjugate). In another preferred embodiment, the non-ester containinglinker moiety is an amido linker moiety (i.e., a PEG-A-DAA conjugate).In yet another preferred embodiment, the non-ester containing linkermoiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

In particular embodiments, the PEG-lipid conjugate is selected from:

The PEG-DAA conjugates are synthesized using standard techniques andreagents known to those of skill in the art. It will be recognized thatthe PEG-DAA conjugates will contain various amide, amine, ether, thio,carbamate, and urea linkages. Those of skill in the art will recognizethat methods and reagents for forming these bonds are well known andreadily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); andFurniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed.(Longman 1989). It will also be appreciated that any functional groupspresent may require protection and deprotection at different points inthe synthesis of the PEG-DAA conjugates. Those of skill in the art willrecognize that such techniques are well known. See, e.g., Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C₁₀)conjugate, a PEG-dilauryloxypropyl (C₁₂) conjugate, aPEG-dimyristyloxypropyl (C₁₄) conjugate, a PEG-dipalmityloxypropyl (C₁₆)conjugate, or a PEG-distearyloxypropyl (C₁₈) conjugate. In theseembodiments, the PEG preferably has an average molecular weight of about750 or about 2,000 daltons. In one particularly preferred embodiment,the PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the “2000”denotes the average molecular weight of the PEG, the “C” denotes acarbamate linker moiety, and the “DMA” denotes dimyristyloxypropyl. Inanother particularly preferred embodiment, the PEG-lipid conjugatecomprises PEG750-C-DMA, wherein the “750” denotes the average molecularweight of the PEG, the “C” denotes a carbamate linker moiety, and the“DMA” denotes dimyristyloxypropyl. In particular embodiments, theterminal hydroxyl group of the PEG is substituted with a methyl group.Those of skill in the art will readily appreciate that otherdialkyloxypropyls can be used in the PEG-DAA conjugates of the presentinvention.

In addition to the foregoing, it will be readily apparent to those ofskill in the art that other hydrophilic polymers can be used in place ofPEG. Examples of suitable polymers that can be used in place of PEGinclude, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

In addition to the foregoing components, the lipid particles (e.g., LNP)of the present invention can further comprise cationic poly(ethyleneglycol) (PEG) lipids or CPLs (see, e.g., Chen et al., Bioconj. Chem.,11:433-437 (2000); U.S. Pat. No. 6,852,334; PCT Publication No. WO00/62813, the disclosures of which are herein incorporated by referencein their entirety for all purposes).

Suitable CPLs include compounds of Formula VI:

A-W—Y  (VI),

wherein A, W, and Y are as described below.

With reference to Formula VI, “A” is a lipid moiety such as anamphipathic lipid, a neutral lipid, or a hydrophobic lipid that acts asa lipid anchor. Suitable lipid examples include, but are not limited to,diacylglycerolyls, dialkylglycerolyls, N—N-dialkylaminos,1,2-diacyloxy-3-aminopropanes, and 1,2-dialkyl-3-aminopropanes.

“W” is a polymer or an oligomer such as a hydrophilic polymer oroligomer. Preferably, the hydrophilic polymer is a biocompatable polymerthat is nonimmunogenic or possesses low inherent immunogenicity.Alternatively, the hydrophilic polymer can be weakly antigenic if usedwith appropriate adjuvants. Suitable nonimmunogenic polymers include,but are not limited to, PEG, polyamides, polylactic acid, polyglycolicacid, polylactic acid/polyglycolic acid copolymers, and combinationsthereof. In a preferred embodiment, the polymer has a molecular weightof from about 250 to about 7,000 daltons.

“Y” is a polycationic moiety. The term polycationic moiety refers to acompound, derivative, or functional group having a positive charge,preferably at least 2 positive charges at a selected pH, preferablyphysiological pH. Suitable polycationic moieties include basic aminoacids and their derivatives such as arginine, asparagine, glutamine,lysine, and histidine; spermine; spermidine; cationic dendrimers;polyamines; polyamine sugars; and amino polysaccharides. Thepolycationic moieties can be linear, such as linear tetralysine,branched or dendrimeric in structure. Polycationic moieties have betweenabout 2 to about 15 positive charges, preferably between about 2 toabout 12 positive charges, and more preferably between about 2 to about8 positive charges at selected pH values. The selection of whichpolycationic moiety to employ may be determined by the type of particleapplication which is desired.

The charges on the polycationic moieties can be either distributedaround the entire particle moiety, or alternatively, they can be adiscrete concentration of charge density in one particular area of theparticle moiety e.g., a charge spike. If the charge density isdistributed on the particle, the charge density can be equallydistributed or unequally distributed. All variations of chargedistribution of the polycationic moiety are encompassed by the presentinvention.

The lipid “A” and the nonimmunogenic polymer “W” can be attached byvarious methods and preferably by covalent attachment. Methods known tothose of skill in the art can be used for the covalent attachment of “A”and “W.” Suitable linkages include, but are not limited to, amide,amine, carboxyl, carbonate, carbamate, ester, and hydrazone linkages. Itwill be apparent to those skilled in the art that “A” and “W” must havecomplementary functional groups to effectuate the linkage. The reactionof these two groups, one on the lipid and the other on the polymer, willprovide the desired linkage. For example, when the lipid is adiacylglycerol and the terminal hydroxyl is activated, for instance withNHS and DCC, to form an active ester, and is then reacted with a polymerwhich contains an amino group, such as with a polyamide (see, e.g., U.S.Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which are hereinincorporated by reference in their entirety for all purposes), an amidebond will form between the two groups.

In certain instances, the polycationic moiety can have a ligandattached, such as a targeting ligand or a chelating moiety forcomplexing calcium. Preferably, after the ligand is attached, thecationic moiety maintains a positive charge. In certain instances, theligand that is attached has a positive charge. Suitable ligands include,but are not limited to, a compound or device with a reactive functionalgroup and include lipids, amphipathic lipids, carrier compounds,bioaffinity compounds, biomaterials, biopolymers, biomedical devices,analytically detectable compounds, therapeutically active compounds,enzymes, peptides, proteins, antibodies, immune stimulators,radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, other targeting moieties, or toxins.

In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % toabout 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8mol % to about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, fromabout 0.9 mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol%, from about 1 mol % to about 1.7 mol %, from about 1.2 mol % to about1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol %to about 1.6 mol %, or from about 1.4 mol % to about 1.5 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle.

In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % toabout 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol %to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5mol % to about 12 mol %, or about 2 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle.

In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol%, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol% (or any fraction thereof or range therein) of the total lipid presentin the particle.

Additional examples, percentages, and/or ranges of lipid conjugatessuitable for use in the lipid particles of the present invention aredescribed in, e.g., PCT Publication No. WO 09/127060, and PCTPublication No. WO 2010/006282, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of lipid conjugate (e.g.,PEG-lipid) present in the lipid particles of the invention is a targetamount, and that the actual amount of lipid conjugate present in theformulation may vary, for example, by ±2 mol %. For example, in the 1:57lipid particle (e.g., LNP) formulation, the target amount of lipidconjugate is 1.4 mol %, but the actual amount of lipid conjugate may be±0.5 mol %, ±0.4 mol %, ±0.3 mol %, 0.2 mol %, ±0.1 mol %, or ±0.05 mol% of that target amount, with the balance of the formulation being madeup of other lipid components (adding up to 100 mol % of total lipidspresent in the particle). Similarly, in the 7:54 lipid particle (e.g.,LNP) formulation, the target amount of lipid conjugate is 6.76 mol %,but the actual amount of lipid conjugate may be ±2 mol %, ±1.5 mol %, ±1mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle).

One of ordinary skill in the art will appreciate that the concentrationof the lipid conjugate can be varied depending on the lipid conjugateemployed and the rate at which the lipid particle is to becomefusogenic.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe lipid particle and, in turn, the rate at which the lipid particlebecomes fusogenic. For instance, when a PEG-DAA conjugate is used as thelipid conjugate, the rate at which the lipid particle becomes fusogeniccan be varied, for example, by varying the concentration of the lipidconjugate, by varying the molecular weight of the PEG, or by varying thechain length and degree of saturation of the alkyl groups on the PEG-DAAconjugate. In addition, other variables including, for example, pH,temperature, ionic strength, etc. can be used to vary and/or control therate at which the lipid particle becomes fusogenic. Other methods whichcan be used to control the rate at which the lipid particle becomesfusogenic will become apparent to those of skill in the art upon readingthis disclosure. Also, by controlling the composition and concentrationof the lipid conjugate, one can control the lipid particle (e.g., LNP)size.

VI. Preparation of Lipid Particles

The lipid particles of the present invention, e.g., LNP, in which anactive agent or therapeutic agent such as an interfering RNA (e.g.,siRNA) is entrapped within the lipid portion of the particle and isprotected from degradation, can be formed by any method known in the artincluding, but not limited to, a continuous mixing method, a directdilution process, and an in-line dilution process.

In particular embodiments, the cationic lipids may comprise lipids ofFormula I or salts thereof, alone or in combination with other cationiclipids. In other embodiments, the non-cationic lipids are eggsphingomyelin (ESM), distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC),1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),dipalmitoyl-phosphatidylcholine (DPPC),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE(1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 trans PE(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls), cholesterol,derivatives thereof, or combinations thereof.

In certain embodiments, the present invention provides nucleicacid-lipid particles (e.g., LNP) produced via a continuous mixingmethod, e.g., a process that includes providing an aqueous solutioncomprising a nucleic acid (e.g., interfering RNA) in a first reservoir,providing an organic lipid solution in a second reservoir (wherein thelipids present in the organic lipid solution are solubilized in anorganic solvent, e.g., a lower alkanol such as ethanol), and mixing theaqueous solution with the organic lipid solution such that the organiclipid solution mixes with the aqueous solution so as to substantiallyinstantaneously produce a lipid vesicle (e.g., liposome) encapsulatingthe nucleic acid within the lipid vesicle. This process and theapparatus for carrying out this process are described in detail in U.S.Patent Application Publication No. 2004/0142025, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a lipid vesicle substantially instantaneously upon mixing. Asused herein, the phrase “continuously diluting a lipid solution with abuffer solution” (and variations) generally means that the lipidsolution is diluted sufficiently rapidly in a hydration process withsufficient force to effectuate vesicle generation. By mixing the aqueoussolution comprising a nucleic acid with the organic lipid solution, theorganic lipid solution undergoes a continuous stepwise dilution in thepresence of the buffer solution (i.e., aqueous solution) to produce anucleic acid-lipid particle.

The nucleic acid-lipid particles formed using the continuous mixingmethod typically have a size of from about 30 nm to about 150 nm, fromabout 40 nm to about 150 nm, from about 50 nm to about 150 nm, fromabout 60 nm to about 130 nm, from about 70 nm to about 110 nm, fromabout 70 nm to about 100 nm, from about 80 nm to about 100 nm, fromabout 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, or 150 nm (or any fraction thereof or range therein). Theparticles thus formed do not aggregate and are optionally sized toachieve a uniform particle size.

In another embodiment, the present invention provides nucleic acid-lipidparticles (e.g., LNP) produced via a direct dilution process thatincludes forming a lipid vesicle (e.g., liposome) solution andimmediately and directly introducing the lipid vesicle solution into acollection vessel containing a controlled amount of dilution buffer. Inpreferred aspects, the collection vessel includes one or more elementsconfigured to stir the contents of the collection vessel to facilitatedilution. In one aspect, the amount of dilution buffer present in thecollection vessel is substantially equal to the volume of lipid vesiclesolution introduced thereto. As a non-limiting example, a lipid vesiclesolution in 45% ethanol when introduced into the collection vesselcontaining an equal volume of dilution buffer will advantageously yieldsmaller particles.

In yet another embodiment, the present invention provides nucleicacid-lipid particles (e.g., LNP) produced via an in-line dilutionprocess in which a third reservoir containing dilution buffer is fluidlycoupled to a second mixing region. In this embodiment, the lipid vesicle(e.g., liposome) solution formed in a first mixing region is immediatelyand directly mixed with dilution buffer in the second mixing region. Inpreferred aspects, the second mixing region includes a T-connectorarranged so that the lipid vesicle solution and the dilution bufferflows meet as opposing 180° flows; however, connectors providingshallower angles can be used, e.g., from about 27° to about 180° (e.g.,about 90°). A pump mechanism delivers a controllable flow of buffer tothe second mixing region. In one aspect, the flow rate of dilutionbuffer provided to the second mixing region is controlled to besubstantially equal to the flow rate of lipid vesicle solutionintroduced thereto from the first mixing region. This embodimentadvantageously allows for more control of the flow of dilution buffermixing with the lipid vesicle solution in the second mixing region, andtherefore also the concentration of lipid vesicle solution in bufferthroughout the second mixing process. Such control of the dilutionbuffer flow rate advantageously allows for small particle size formationat reduced concentrations.

These processes and the apparatuses for carrying out these directdilution and in-line dilution processes are described in detail in U.S.Patent Application Publication No. 2007/0042031, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

The nucleic acid-lipid particles formed using the direct dilution andin-line dilution processes typically have a size of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, from about 70 nm to about 100 nm, from about 80 nm toabout 100 nm, from about 90 nm to about 100 nm, from about 70 to about90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm,less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm,35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135 nm, 140 nm, 145 nm, or 150 nm (or any fraction thereof or rangetherein). The particles thus formed do not aggregate and are optionallysized to achieve a uniform particle size.

If needed, the lipid particles of the invention (e.g., LNP) can be sizedby any of the methods available for sizing liposomes. The sizing may beconducted in order to achieve a desired size range and relatively narrowdistribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles, is described in U.S. Pat. No. 4,737,323, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Sonicating a particle suspension either by bath orprobe sonication produces a progressive size reduction down to particlesof less than about 50 nm in size. Homogenization is another method whichrelies on shearing energy to fragment larger particles into smallerones. In a typical homogenization procedure, particles are recirculatedthrough a standard emulsion homogenizer until selected particle sizes,typically between about 60 and about 80 nm, are observed. In bothmethods, the particle size distribution can be monitored by conventionallaser-beam particle size discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In some embodiments, the nucleic acids present in the particles areprecondensed as described in, e.g., U.S. patent application Ser. No.09/744,103, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

In other embodiments, the methods may further comprise adding non-lipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable non-lipid polycationsinclude, hexadimethrine bromide (sold under the brand name POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine, and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In some embodiments, the nucleic acid to lipid ratios (mass/mass ratios)in a formed nucleic acid-lipid particle (e.g., LNP) will range fromabout 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about0.08. The ratio of the starting materials (input) also falls within thisrange. In other embodiments, the particle preparation uses about 400 μgnucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratioof about 0.01 to about 0.08 and, more preferably, about 0.04, whichcorresponds to 1.25 mg of total lipid per 50 μg of nucleic acid. Inother preferred embodiments, the particle has a nucleic acid:lipid massratio of about 0.08.

In other embodiments, the lipid to nucleic acid ratios (mass/massratios) in a formed nucleic acid-lipid particle (e.g., LNP) will rangefrom about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100(100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) toabout 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4(4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), fromabout 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1),from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25(25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) toabout 20 (20:1), from about 5 (5:1) to about 15 (15:1), from about 5(5:1) to about 10 (10:1), or about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9(9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15 (15:1),16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21 (21:1), 22(22:1), 23 (23:1), 24 (24:1), or 25 (25:1), or any fraction thereof orrange therein. The ratio of the starting materials (input) also fallswithin this range.

As previously discussed, the conjugated lipid may further include a CPL.A variety of general methods for making LNP-CPLs (CPL-containing LNP)are discussed herein. Two general techniques include the“post-insertion” technique, that is, insertion of a CPL into, forexample, a pre-formed LNP, and the “standard” technique, wherein the CPLis included in the lipid mixture during, for example, the LNP formationsteps. The post-insertion technique results in LNP having CPLs mainly inthe external face of the LNP bilayer membrane, whereas standardtechniques provide LNP having CPLs on both internal and external faces.The method is especially useful for vesicles made from phospholipids(which can contain cholesterol) and also for vesicles containingPEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of making LNP-CPLsare taught, for example, in U.S. Pat. Nos. 5,705,385; 6,586,410;5,981,501; 6,534,484; and 6,852,334; U.S. Patent Application PublicationNo. 2002/0072121; and PCT Publication No. WO 00/62813, the disclosuresof which are herein incorporated by reference in their entirety for allpurposes.

VII. Kits

The present invention also provides lipid particles (e.g., LNP) in kitform. In some embodiments, the kit comprises a container which iscompartmentalized for holding the various elements of the lipidparticles (e.g., the active agents or therapeutic agents such as nucleicacids and the individual lipid components of the particles). Preferably,the kit comprises a container (e.g., a vial or ampoule) which holds thelipid particles of the invention (e.g., LNP), wherein the particles areproduced by one of the processes set forth herein. In certainembodiments, the kit may further comprise an endosomal membranedestabilizer (e.g., calcium ions). The kit typically contains theparticle compositions of the invention, either as a suspension in apharmaceutically acceptable carrier or in dehydrated form, withinstructions for their rehydration (if lyophilized) and administration.

The lipid particles of the present invention can be tailored topreferentially target particular tissues, organs, or tumors of interest.In some instances, the 1:57 lipid particle (e.g., LNP) formulation canbe used to preferentially target the liver (e.g., normal liver tissue).In other instances, the 7:54 lipid particle (e.g., LNP) formulation canbe used to preferentially target solid tumors such as liver tumors andtumors outside of the liver. In preferred embodiments, the kits of theinvention comprise these liver-directed and/or tumor-directed lipidparticles, wherein the particles are present in a container as asuspension or in dehydrated form.

In certain other instances, it may be desirable to have a targetingmoiety attached to the surface of the lipid particle to further enhancethe targeting of the particle. Methods of attaching targeting moieties(e.g., antibodies, proteins, etc.) to lipids (such as those used in thepresent particles) are known to those of skill in the art.

VIII. Administration of Lipid Particles

Once formed, the lipid particles of the invention (e.g., LNP) are usefulfor the introduction of active agents or therapeutic agents (e.g.,nucleic acids such as interfering RNA) into cells. Accordingly, thepresent invention also provides methods for introducing an active agentor therapeutic agent such as a nucleic acid (e.g., interfering RNA) intoa cell. In some instances, the cell is a liver cell such as, e.g., ahepatocyte present in liver tissue. In other instances, the cell is atumor cell such as, e.g., a tumor cell present in a solid tumor. Themethods are carried out in vitro or in vivo by first forming theparticles as described above and then contacting the particles with thecells for a period of time sufficient for delivery of the active agentor therapeutic agent to the cells to occur.

The lipid particles of the invention (e.g., LNP) can be adsorbed toalmost any cell type with which they are mixed or contacted. Onceadsorbed, the particles can either be endocytosed by a portion of thecells, exchange lipids with cell membranes, or fuse with the cells.Transfer or incorporation of the active agent or therapeutic agent(e.g., nucleic acid) portion of the particle can take place via any oneof these pathways. In particular, when fusion takes place, the particlemembrane is integrated into the cell membrane and the contents of theparticle combine with the intracellular fluid.

The lipid particles of the invention (e.g., LNP) can be administeredeither alone or in a mixture with a pharmaceutically acceptable carrier(e.g., physiological saline or phosphate buffer) selected in accordancewith the route of administration and standard pharmaceutical practice.Generally, normal buffered saline (e.g., 135-150 mM NaCl) will beemployed as the pharmaceutically acceptable carrier. Other suitablecarriers include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, including glycoproteins for enhanced stability,such as albumin, lipoprotein, globulin, etc. Additional suitablecarriers are described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES,Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As usedherein, “carrier” includes any and all solvents, dispersion media,vehicles, coatings, diluents, antibacterial and antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. The phrase “pharmaceuticallyacceptable” refers to molecular entities and compositions that do notproduce an allergic or similar untoward reaction when administered to ahuman.

The pharmaceutically acceptable carrier is generally added followinglipid particle formation. Thus, after the lipid particle (e.g., LNP) isformed, the particle can be diluted into pharmaceutically acceptablecarriers such as normal buffered saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2 to 5%, to as much as about 10 to 90% by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol, and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

In some embodiments, the lipid particles of the invention (e.g., LNP)are particularly useful in methods for the therapeutic delivery of oneor more nucleic acids comprising an interfering RNA sequence (e.g.,siRNA). In particular, it is an object of this invention to provide invitro and in vivo methods for treatment of a disease or disorder in amammal (e.g., a rodent such as a mouse or a primate such as a human,chimpanzee, or monkey) by downregulating or silencing the transcriptionand/or translation of one or more target nucleic acid sequences or genesof interest. As a non-limiting example, the methods of the invention areuseful for in vivo delivery of interfering RNA (e.g., siRNA) to theliver and/or tumor of a mammalian subject. In certain embodiments, thedisease or disorder is associated with expression and/or overexpressionof a gene and expression or overexpression of the gene is reduced by theinterfering RNA (e.g., siRNA). In certain other embodiments, atherapeutically effective amount of the lipid particle may beadministered to the mammal. In some instances, an interfering RNA (e.g.,siRNA) is formulated into a LNP, and the particles are administered topatients requiring such treatment. In other instances, cells are removedfrom a patient, the interfering RNA is delivered in vitro (e.g., using aLNP described herein), and the cells are reinjected into the patient.

A. In Vivo Administration

Systemic delivery for in vivo therapy, e.g., delivery of a therapeuticnucleic acid to a distal target cell via body systems such as thecirculation, has been achieved using nucleic acid-lipid particles suchas those described in PCT Publication Nos. WO 05/007196, WO 05/121348,WO 05/120152, and WO 04/002453, the disclosures of which are hereinincorporated by reference in their entirety for all purposes. Thepresent invention also provides fully encapsulated lipid particles thatprotect the nucleic acid from nuclease degradation in serum, arenon-immunogenic, are small in size, and are suitable for repeat dosing.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation (e.g.,intransal or intratracheal), transdermal application, or rectaladministration. Administration can be accomplished via single or divideddoses. The pharmaceutical compositions can be administered parenterally,i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.5,286,634). Intracellular nucleic acid delivery has also been discussedin Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino etal., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. DrugCarrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).Still other methods of administering lipid-based therapeutics aredescribed in, for example, U.S. Pat. Nos. 3,993,754; 4,145,410;4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles canbe administered by direct injection at the site of disease or byinjection at a site distal from the site of disease (see, e.g., Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71(1994)). The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

In embodiments where the lipid particles of the present invention (e.g.,LNP) are administered intravenously, at least about 5%, 10%, 15%, 20%,or 25% of the total injected dose of the particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% ofthe total injected dose of the lipid particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In certain instances,more than about 10% of a plurality of the particles is present in theplasma of a mammal about 1 hour after administration. In certain otherinstances, the presence of the lipid particles is detectable at leastabout 1 hour after administration of the particle. In certainembodiments, the presence of a therapeutic agent such as a nucleic acidis detectable in cells of the lung, liver, tumor, or at a site ofinflammation at about 8, 12, 24, 36, 48, 60, 72 or 96 hours afteradministration. In other embodiments, downregulation of expression of atarget sequence by an interfering RNA (e.g., siRNA) is detectable atabout 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration. In yetother embodiments, downregulation of expression of a target sequence byan interfering RNA (e.g., siRNA) occurs preferentially in liver cells(e.g., hepatocytes), tumor cells, or in cells at a site of inflammation.In further embodiments, the presence or effect of an interfering RNA(e.g., siRNA) in cells at a site proximal or distal to the site ofadministration or in cells of the lung, liver, or a tumor is detectableat about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16,18, 19, 20, 22, 24, 26, or 28 days after administration. In additionalembodiments, the lipid particles (e.g., LNP) of the invention areadministered parenterally or intraperitoneally.

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering nucleic acid compositions directly tothe lungs via nasal aerosol sprays have been described, e.g., in U.S.Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins and lysophosphatidyl-glycerol compounds(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceuticalarts. Similarly, transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045. The disclosures of the above-described patents are hereinincorporated by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions are preferablyadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically, or intrathecally.

Generally, when administered intravenously, the lipid particleformulations are formulated with a suitable pharmaceutical carrier. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found, for example, in REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may bedelivered via oral administration to the individual. The particles maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes). These oral dosage forms may also contain thefollowing: binders, gelatin; excipients, lubricants, and/or flavoringagents. When the unit dosage form is a capsule, it may contain, inaddition to the materials described above, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Of course, any material used inpreparing any unit dosage form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed.

Typically, these oral formulations may contain at least about 0.1% ofthe lipid particles or more, although the percentage of the particlesmay, of course, be varied and may conveniently be between about 1% or 2%and about 60% or 70% or more of the weight or volume of the totalformulation. Naturally, the amount of particles in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

Formulations suitable for oral administration can consist of: (a) liquidsolutions, such as an effective amount of a packaged therapeutic agentsuch as nucleic acid (e.g., interfering RNA) suspended in diluents suchas water, saline, or PEG 400; (b) capsules, sachets, or tablets, eachcontaining a predetermined amount of a therapeutic agent such as nucleicacid (e.g., interfering RNA), as liquids, solids, granules, or gelatin;(c) suspensions in an appropriate liquid; and (d) suitable emulsions.Tablet forms can include one or more of lactose, sucrose, mannitol,sorbitol, calcium phosphates, corn starch, potato starch,microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc,magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically compatible carriers. Lozenge forms can comprise atherapeutic agent such as nucleic acid (e.g., interfering RNA) in aflavor, e.g., sucrose, as well as pastilles comprising the therapeuticagent in an inert base, such as gelatin and glycerin or sucrose andacacia emulsions, gels, and the like containing, in addition to thetherapeutic agent, carriers known in the art.

In another example of their use, lipid particles can be incorporatedinto a broad range of topical dosage forms. For instance, a suspensioncontaining nucleic acid-lipid particles such as LNP can be formulatedand administered as gels, oils, emulsions, topical creams, pastes,ointments, lotions, foams, mousses, and the like.

When preparing pharmaceutical preparations of the lipid particles of theinvention, it is preferable to use quantities of the particles whichhave been purified to reduce or eliminate empty particles or particleswith therapeutic agents such as nucleic acid associated with theexternal surface.

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as primates(e.g., humans and chimpanzees as well as other nonhuman primates),canines, felines, equines, bovines, ovines, caprines, rodents (e.g.,rats and mice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio oftherapeutic agent (e.g., nucleic acid) to lipid, the particulartherapeutic agent (e.g., nucleic acid) used, the disease or disorderbeing treated, the age, weight, and condition of the patient, and thejudgment of the clinician, but will generally be between about 0.01 andabout 50 mg per kilogram of body weight, preferably between about 0.1and about 5 mg/kg of body weight, or about 10⁸-10¹⁰ particles peradministration (e.g., injection).

B. In Vitro Administration

For in vitro applications, the delivery of therapeutic agents such asnucleic acids (e.g., interfering RNA) can be to any cell grown inculture, whether of plant or animal origin, vertebrate or invertebrate,and of any tissue or type. In preferred embodiments, the cells areanimal cells, more preferably mammalian cells, and most preferably humancells (e.g., tumor cells or hepatocytes).

Contact between the cells and the lipid particles, when carried out invitro, takes place in a biologically compatible medium. Theconcentration of particles varies widely depending on the particularapplication, but is generally between about 1 μmol and about 10 mmol.Treatment of the cells with the lipid particles is generally carried outat physiological temperatures (about 37° C.) for periods of time of fromabout 1 to 48 hours, preferably of from about 2 to 4 hours.

In one group of preferred embodiments, a lipid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/ml, more preferably about 2×10⁴ cells/ml.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 0.2 μg/ml, more preferably about 0.1 μg/ml.

To the extent that tissue culture of cells may be required, it iswell-known in the art. For example, Freshney, Culture of Animal Cells, aManual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchleret al., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977), and the references cited thereinprovide a general guide to the culture of cells. Cultured cell systemsoften will be in the form of monolayers of cells, although cellsuspensions are also used.

Using an Endosomal Release Parameter (ERP) assay, the deliveryefficiency of the LNP or other lipid particle of the invention can beoptimized. An ERP assay is described in detail in U.S. PatentApplication Publication No. 2003/0077829, the disclosure of which isherein incorporated by reference in its entirety for all purposes. Moreparticularly, the purpose of an ERP assay is to distinguish the effectof various cationic lipids and helper lipid components of LNP or otherlipid particle based on their relative effect on binding/uptake orfusion with/destabilization of the endosomal membrane. This assay allowsone to determine quantitatively how each component of the LNP or otherlipid particle affects delivery efficiency, thereby optimizing the LNPor other lipid particle. Usually, an ERP assay measures expression of areporter protein (e.g., luciferase, β-galactosidase, green fluorescentprotein (GFP), etc.), and in some instances, a LNP formulation optimizedfor an expression plasmid will also be appropriate for encapsulating aninterfering RNA. In other instances, an ERP assay can be adapted tomeasure downregulation of transcription or translation of a targetsequence in the presence or absence of an interfering RNA (e.g., siRNA).By comparing the ERPs for each of the various LNP or other lipidparticles, one can readily determine the optimized system, e.g., the LNPor other lipid particle that has the greatest uptake in the cell.

C. Cells for Delivery of Lipid Particles

The compositions and methods of the present invention are used to treata wide variety of cell types, in vivo and in vitro. Suitable cellsinclude, but are not limited to, hepatocytes, reticuloendothelial cells(e.g., monocytes, macrophages, etc.), fibroblast cells, endothelialcells, platelet cells, other cell types infected and/or susceptible ofbeing infected with viruses, hematopoietic precursor (stem) cells,keratinocytes, skeletal and smooth muscle cells, osteoblasts, neurons,quiescent lymphocytes, terminally differentiated cells, slow ornoncycling primary cells, parenchymal cells, lymphoid cells, epithelialcells, bone cells, and the like.

In particular embodiments, an active agent or therapeutic agent such asa nucleic acid (e.g., an interfering RNA) is delivered to cancer cells(e.g., cells of a solid tumor) including, but not limited to, livercancer cells, lung cancer cells, colon cancer cells, rectal cancercells, anal cancer cells, bile duct cancer cells, small intestine cancercells, stomach (gastric) cancer cells, esophageal cancer cells,gallbladder cancer cells, pancreatic cancer cells, appendix cancercells, breast cancer cells, ovarian cancer cells, cervical cancer cells,prostate cancer cells, renal cancer cells, cancer cells of the centralnervous system, glioblastoma tumor cells, skin cancer cells, lymphomacells, choriocarcinoma tumor cells, head and neck cancer cells,osteogenic sarcoma tumor cells, and blood cancer cells.

In vivo delivery of lipid particles such as LNP encapsulating a nucleicacid (e.g., an interfering RNA) is suited for targeting cells of anycell type. The methods and compositions can be employed with cells of awide variety of vertebrates, including mammals, such as, e.g, canines,felines, equines, bovines, ovines, caprines, rodents (e.g., mice, rats,and guinea pigs), lagomorphs, swine, and primates (e.g. monkeys,chimpanzees, and humans).

D. Detection of Lipid Particles

In some embodiments, the lipid particles of the present invention (e.g.,LNP) are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 ormore hours. In other embodiments, the lipid particles of the presentinvention (e.g., LNP) are detectable in the subject at about 8, 12, 24,48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24,25, or 28 days after administration of the particles. The presence ofthe particles can be detected in the cells, tissues, or other biologicalsamples from the subject. The particles may be detected, e.g., by directdetection of the particles, detection of a therapeutic nucleic acid suchas an interfering RNA (e.g., siRNA) sequence, detection of the targetsequence of interest (i.e., by detecting expression or reducedexpression of the sequence of interest), or a combination thereof.

1. Detection of Particles

Lipid particles of the invention such as LNP can be detected using anymethod known in the art. For example, a label can be coupled directly orindirectly to a component of the lipid particle using methods well-knownin the art. A wide variety of labels can be used, with the choice oflabel depending on sensitivity required, ease of conjugation with thelipid particle component, stability requirements, and availableinstrumentation and disposal provisions. Suitable labels include, butare not limited to, spectral labels such as fluorescent dyes (e.g.,fluorescein and derivatives, such as fluorescein isothiocyanate (FITC)and Oregon Green™; rhodamine and derivatives such Texas red,tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin,phycoerythrin, AMCA, CyDyes™, and the like; radiolabels such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes such as horse radish peroxidase,alkaline phosphatase, etc.; spectral colorimetric labels such ascolloidal gold or colored glass or plastic beads such as polystyrene,polypropylene, latex, etc. The label can be detected using any meansknown in the art.

2. Detection of Nucleic Acids

Nucleic acids (e.g., interfering RNA) are detected and quantified hereinby any of a number of means well-known to those of skill in the art. Thedetection of nucleic acids may proceed by well-known methods such asSouthern analysis, Northern analysis, gel electrophoresis, PCR,radiolabeling, scintillation counting, and affinity chromatography.Additional analytic biochemical methods such as spectrophotometry,radiography, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), and hyperdiffusion chromatography may also be employed.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in, e.g., “Nucleic Acid Hybridization, A PracticalApproach,” Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of the hybridization assays may be enhanced through theuse of a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification, and otherRNA polymerase mediated techniques (e.g., NASBA™) are found in Sambrooket al., In Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS INMOLECULAR BIOLOGY, eds., Current Protocols, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (2002); as well as U.S.Pat. No. 4,683,202; PCR Protocols, A Guide to Methods and Applications(Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990);Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research,3:81 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell etal., J Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077(1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene,4:560 (1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan andMalek, Biotechnology, 13:563 (1995). Improved methods of cloning invitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039.Other methods described in the art are the nucleic acid sequence basedamplification (NASBA™, Cangene, Mississauga, Ontario) and Qβ-replicasesystems. These systems can be used to directly identify mutants wherethe PCR or LCR primers are designed to be extended or ligated only whena select sequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation. The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

Nucleic acids for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage et al.,Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an automatedsynthesizer, as described in Needham VanDevanter et al., Nucleic AcidsRes., 12:6159 (1984). Purification of polynucleotides, where necessary,is typically performed by either native acrylamide gel electrophoresisor by anion exchange HPLC as described in Pearson et al., J. Chrom.,255:137 149 (1983). The sequence of the synthetic polynucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology, 65:499.

An alternative means for determining the level of transcription is insitu hybridization. In situ hybridization assays are well-known and aregenerally described in Angerer et al., Methods Enzymol., 152:649 (1987).In an in situ hybridization assay, cells are fixed to a solid support,typically a glass slide. If DNA is to be probed, the cells are denaturedwith heat or alkali. The cells are then contacted with a hybridizationsolution at a moderate temperature to permit annealing of specificprobes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

IX. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

General Methods:

All reactions were carried out at room temperature under a positivepressure of nitrogen unless otherwise stated. All reagents werepurchased from commercial sources and used without further purification.Reaction progress was monitored by TLC on silica gel 60 F254 (0.25 mm,E. Merck). Spots were detected under UV light or by charring withanisaldehyde or copper sulphate stains. All column chromatography wascarried out on silica gel 60 (40-60 μM). The ratio between silica geland crude product ranged from 100 to 50:1. ¹H NMR spectra were recordedat 300 MHz or 400 MHz and chemical shifts were internally referenced tothe residual protonated solvent (7.27 ppm CHCl₃). Organic solutions wereconcentrated under vacuum at <40° C.

Example 1

This Example describes the synthesis of exemplary, trialkyl, cationiclipids of the present invention.

Synthetic Scheme for Compound 9

Synthesis of Compound 2

To a cooled solution (0° C.) of (Z)-dec-4-en-1-ol 9 (20 g, 128.0 mmol)and triethylamine (26.7 mL, 191.9 mmol) in anhydrous dichloromethane(200 mL) was slowly added methane sulfonyl chloride (14.9 mL, 191.9mmol). The solution was stirred for 30 min at room temperature thendiluted with dichloromethane (100 mL) The solution was washed withsaturated sodium bicarbonate (3×150 mL) and then the combined aqueouswashes were extracted with dichloromethane (150 mL). The combineddichloromethane extracts were dried on magnesium sulfate, filtered andconcentrated in vacuo to dryness. The residue was filtered through a padof silica (100% dichloromethane) to afford (Z)-dec-4-enylmethanesulfonate 2 as a yellow oil (28.5 g, 95%). Rf 0.5 (100% CH₂Cl₂).

Synthesis of Compound 3

To a solution of (Z)-dec-4-enyl methanesulfonate 2 (28.5 g, 121.1 mmol)in 2-methyltetrahydrofuran (280 mL) was added tetrabutylammonium bromide(48.8 g, 151.4 mmol). The solution was stirred at 80° C. for 30 minutesunder nitrogen, then diluted with ether (150 mL) and washed with water(75 mL) and brine (75 mL). The ether solution was dried on magnesiumsulfate, filtered and concentrated in vacuo to dryness. The pale yellowoil was filtered through a pad of silica (100% hexanes) to afford(Z)-1-bromodec-4-ene 3 as a colorless oil (23.0 g, 87%). Rf 0.9 (10%EtOAc-Hexanes).

Synthesis of Compound 4

To a suspension of magnesium turnings (1.4 g, 55.1 mmol) in anhydrousTHF (6 mL) under nitrogen was slowly added a solution of(Z)-1-bromodec-4-ene 3 (11.5 g, 52.5 mmol) in THF (12 mL). The reactionmixture was stirred at 45° C. for 30 minutes under nitrogen. Thesolution was cooled to 0° C. and a solution of ethyl formate (4.1 g,55.1 mmol) in THF (12 mL) was added dropwise to over 5 minutes. Thesolution was stirred at room temperature for 2 hours then cooled to −15°C. and quenched slowly with water (10 mL) followed by 5M hydrochloricacid (15 mL). Once the magnesium had completely dissolved, the solutionwas diluted with water (50 mL) and extracted with hexanes (3×75 mL). Thecombined extracts were dried on magnesium sulfate, filtered andconcentrated in vacuo to dryness. The residue obtained was dissolved inethanol (40 mL) and a solution of potassium hydroxide (4.4 g, 78.7mmol)) in water (10 mL) was added. The reaction mixture was stirredvigorously for 30 minutes then concentrated in vacuo to remove ethanol.The solution was then made acidic with 5M hydrochloric acid (15 mL) andextracted with hexanes (3×75 mL). The combined hexanes extracts weredried on magnesium sulfate, filtered and concentrated in vacuo todryness. The crude product was purified by column chromatography (100%hexanes to 2.5% ethyl acetate in hexanes) to afford(6Z,15Z)-henicosa-6,15-dien-11-ol 4 as a pale yellow oil (5.6 g, 35%).Rf 0.4 (10% EtOAc-Hexanes).

Synthesis of Compound 5

To a cooled solution (0° C.) of 6Z,15Z)-henicosa-6,15-lien-11-ol 4 (5.6g, 18.2 mmol) and triethylamine (3.8 mL, 27.2 mmol) in anhydrousdichloromethane (50 mL) was slowly added methanesulfonyl chloride (2.1mL, 27.2 mmol). The reaction mixture was stirred for 2 hours at roomtemperature then diluted with dichloromethane (50 mL). The solution waswashed with saturated sodium bicarbonate (3×25 mL) then the combinedaqueous washes were extracted with dichloromethane (50 mL). The combineddichloromethane extracts were dried on magnesium sulfate, filtered andconcentrated in vacuo to dryness. The pale yellow oil was filteredthrough a pad of silica (100% DCM) to afford(6Z,15Z)-henicosa-6,15-dien-11-yl methanesulfonate 5 as a crudecolorless oil (7.6 g). Rf 0.8 (100% CH₂Cl₂).

Synthesis of Compound 6

A solution of (6Z,15Z)-henicosa-6,15-dien-11-yl methanesulfonate 5 (7.6g, 19.6 mmol) and sodium cyanide (4.8 g, 98.1 mmol) in anhydrous DMF (60mL) was heated to 60° C. overnight. Upon completion, the reactionmixture was poured into water (200 mL) and extracted with ethyl acetate(3×100 mL). The combine ethyl acetate extracts were washed with brine(3×100 mL), dried on magnesium sulfate, filtered and concentrated invacuo to dryness. The product was purified by column chromatography(100% Hexanes to 1% ethyl acetate in hexanes) to afford(Z)-2-((Z)-dec-4-enyl)dodec-6-enenitrile 6 as a colorless oil (6.6 g,97%). Rf 0.75 (10% EtOAc-Hexanes).

Synthesis of Compound 7

To a cooled solution (−78° C.) of(Z)-2-((Z)-dec-4-enyl)dodec-6-enenitrile 6 (4.0 g, 12.6 mmol) inanhydrous dichloromethane (125 mL) was added slowly a 1M solution ofdiisobutylaluminum hydride in hexanes (5.6 mL, 31.5 mmol). The solutionwas warmed to −15° C. and stirred for 1 hour. Upon completion, thereaction was quenched with 5% hydrochloric acid (30 mL) and stirred at−15° C. until the evolution of hydrogen gas ceased. The solution wasthen diluted with dichloromethane (75 mL) and the organic layer waswashed with 5M hydrochloric acid (100 mL). The dichloromethane extractswere dried on magnesium sulfate, filtered and concentrated in vacuo todryness. The residue was purified by column chromatography (100% Hexanesto 2% ethyl acetate in hexanes) to afford(Z)-2-((Z)-dec-4-enyl)dodec-6-enal 7 as a colorless oil (3.9 g, 97%). Rf0.65 (5% EtOAc-Hexanes).

Synthesis of Compound 8

To a suspension of magnesium turnings (0.6 g, 23.9 mmol) intetrahydrofuran (5 mL) was slowly added a solution of(Z)-1-bromodec-4-ene 3 (4.5 g, 20.5 mmol) in tetrahydrofuran (5 mL). Thereaction mixture was stirred for 30 minutes at room temperature then asolution of (Z)-2-((Z)-dec-4-enyl)dodec-6-enal 7 in tetrahydrofuran (5mL) was added. The solution was stirred for 15 minutes at roomtemperature then poured into 5% hydrochloric acid (50 mL) and ice (100mL). The solution was extracted with ether (2×150 mL). The combine etherextracts were dried on magnesium sulfate, filtered, and concentrated invacuo to dryness. The residue was purified by column chromatography (1%ethyl acetate in hexanes) to afford(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 8 as a colorless oil(4.8 g, 76%). Rf 0.45 (10% EtOAc-Hexanes).

Synthesis of Compound 9

To a solution of (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 8(0.4 g, 0.9 mmol), 4-(dimethylamino)butanoic acid hydrochloride (0.2 g,1.3 mmol), EDCI hydrochloride (0.25 g, 1.3 mmol), diisopropylethylamine(0.4 mL, 2.6 mmol) in anhydrous dichloromethane (10 mL) was addeddimethylaminopyridine (5 mg). The solution was refluxed for 2 hours thenstirred at room temperature for 2 hours. The mixture is concentrated invacuo to dryness and purified by column chromatography (100% ethylacetate) to afford 6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl4-(dimethylamino)butanoate 9 as a pale yellow oil. ¹H NMR (400 MHz,CDCl₃) δ 5.36 (m, 6H), 4.93 (m, 1H), 2.30 (m, 4H), 2.22 (s, 6H), 2.03(m, 12H), 1.88 (m, 2H), 1.66-1.18 (m, 31H), 0.90 (m, 9H). Rf 0.3 (10%MeOH—CH₂Cl₂).

Synthetic Scheme for Compounds 11 and 13

Synthesis of Compound 10

To a solution of (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 8(2.4 g, 5.2 mmol), 6-bromohexanoic acid (1.5 g, 7.8 mmol), EDCIhydrochloride (1.5 g, 7.8 mmol), diisopropylethylamine (2.0 g, 15.6mmol) in anhydrous dichloromethane (25 mL) was addeddimethylaminopyridine (15 mg). The solution was refluxed for 2 hours,cooled to room temperature and concentrated in vacuo to dryness. Thereaction mixture was purified by column chromatography on silica gel 60(2″ W×10″ L; eluted with 5% EtOAc/Hex) to afford(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 6-bromohexanoate 10as a colorless oil (3.1 g, 94%). Rf 0.5 (10% EtOAc-Hexanes).

Synthesis of Compound 11

To (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 6-bromohexanoate10 (3.1 g, 4.9 mmol) in a teflon sealed pressure vessel was added 5.6 Mdimethylamine in ethanol (20 mL) and the reaction was heated to 70° C.and stirred overnight. Once complete, the reaction was concentrated invacuo to dryness. The residue was dissolved in ethyl acetate (100 mL)and washed with sodium bicarbonate solution (2×50 mL). The ethyl acetatelayer was dried on magnesium sulfate, filtered, and concentrated invacuo to dryness. The residue was purified by column chromatography(100% EtOAc) to afford(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl6-(dimethylamino)hexanoate 11 as a pale yellow oil (2.0 g, 69%), ¹H NMR(400 MHz, CDCl₃) δ 5.36 (m, 6H), 4.93 (m, 1H), 2.27 (m, 10H), 2.00 (m,12H), 1.63 (m, 6H), 1.51 (m, 6H), 1.28 (m, 25H), 0.90 (m, 9H). Rf 0.3(10% MeOH—CH₂Cl₂).

Synthesis of Compound 12

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 6-bromohexanoate 10,(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 5-bromopentanoate 12was obtained as a colorless oil (3.3 g, 61%) from(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 8 (4.0 g, 8.7 mmol),6-bromo-n-valeric acid (2.4 g, 13.0 mmol), EDCI hydrochloride (2.5 g,13.0 mmol), diisopropylethylamine (3.4 g, 26.0 mmol) anddimethylaminopyridine (10 mg). Rf 0.5 (10% EtOAc-Hexanes).

Synthesis of Compound 13

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl6-(dimethylamino)hexanoate 11,(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl5-(dimethylamino)pentanoate 13 was obtained as a pale yellow oil (1.9 g,62%) from 5.6 M dimethylamine in ethanol (20 mL). ¹H NMR (400 MHz,CDCl₃) δ 5.45-5.28 (m, 6H), 4.95-4.90 (m, 1H), 2.34-2.23 (m, 4H),2.23-2.20 (s, 6H), 2.06-1.92 (m, 12H), 1.70-1.58 (m, 5H), 1.58-1.44 (m,5H), 1.44-1.15 (m, 25H), 0.92-0.87 (m, 9H). Rf 0.4 (10% MeOH—CH₂Cl₂).

Synthetic Scheme for Compound 14

Synthesis of Compound 14

A flask containing(6Z,16Z)-12-((Z)-non-4-en-1-yl)tricosa-6,16-dien-11-yl5-(dimethylamino)pentanoate 13 (200 mg, 0.34 mmol) was evacuated andback-filled with nitrogen (twice) then treated with Pd/C (150 mg, 10%w/w) and subsequently suspended in EtOAc (10 mL). The reaction flask wasthen evacuated and back-filled with H₂ (3×) and the mixture vigorouslystirred (18 h). The H₂ was then evacuated and the flask back-filled withN₂. The reaction mixture was filtered through Celite, rinsing the filtercake with EtOAc, and the filtrate was concentrated. The crude materialwas subjected to chromatography (EtOAc) to yield 12-nonyltricosan-11-yl5-(dimethylamino)pentanoate 14 (100 mg, 50%) as a colorless oil. Rf 0.35(10% CH₃OH—CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃, δ_(H)) 4.95-4.90 (m, 1H),2.31 (t, 2H), 2.27 (t, 2H), 2.21 (s, 6H), 1.68-1.60 (m, 3H), 1.58-1.42(m, 5H), 1.38-1.16 (m, 54H), 0.88 (t, 6H).

Synthetic Scheme for Compounds 19 and 21

Synthesis of Compound 15

Using an analogous procedure to that described for the synthesis of(Z)-dec-4-enyl methanesulfonate 2, (Z)-non-3-enyl methanesulfonate 15was obtained as a yellow oil (28.5 g, 92%) from (Z)-non-3-en-1-ol (20.0g, 128.0 mmol), triethylamine (26.7 mL, 191.9 mmol) and methane sulfonylchloride (14.9 mL, 191.9 mmol). Rf 0.15 (30% Ethyl acetate-hexanes).

Synthesis of Compound 16

Using an analogous procedure to that described for the synthesis of(Z)-1-bromodec-4-ene 3, (Z)-1-bromonon-3-ene 16 was obtained as acolorless oil (27.0 g, quantitative) from (Z)-dec-4-enylmethanesulfonate 15 (28.5 g, 129 mmol) and tetrabutylammonium bromide(52.0 g, 161.4 mmol). Rf 0.6 (10% EtOAc-Hexanes).

Synthesis of Compound 17

A 100 mL round bottom flask was charged with magnesium turnings (0.6 g,25.7 mmol) and a stir bar. The flask was dried with a heat gun for 5minutes. The flask was charged with THF (5 mL) and a single grain oniodine. A solution of (Z)-1-bromonon-3-ene (4.5 g, 22.0 mmol) in THF (5mL) was added slowly to the mixture and reaction was refluxed for 30minutes under nitrogen. The solution was cooled to room temperature anda solution of (Z)-2-((Z)-dec-4-enyl)dodec-6-enal 7 (4.7 g, 14.7 mmol) inTHF (5 mL) was added. The solution was stirred overnight at roomtemperature and upon completion the mixture was poured into 5% HCl (50mL) and ice (100 mL). The solution was extracted with ether (2×150 mL)and the combined ether extracts were dried on magnesium sulfate,filtered and concentrated in vacuo to dryness. The residue was purifiedby column chromatography (column: 2″ W×8″ L; eluted with 100% Hexanes to5% ethyl acetate in hexanes) to afford(6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-ol 17 as a colorlessoil (5.4 g, 82%). Rf 0.5 (10% EtOAc-Hexanes).

Synthesis of Compound 18

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 6-bromohexanoate 10,(6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-yl 5-bromopentanoate18 was obtained as a colorless oil (0.9 g, 66%) from(6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-ol 17 (0.35 g, 0.7mmol), 5-bromo-n-valeric acid (0.60 g, 3.4 mmol), EDCI (0.60 g, 3.4mmol), diisopropylethylamine (0.90 g, 6.7 mmol) and DMAP (5 mg,catalyst). Rf 0.5 (10% EtOAc-Hexanes).

Synthesis of Compound 19

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl6-(dimethylamino)hexanoate 11,(6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-yl5-(dimethylamino)pentanoate 19 was obtained as a colorless oil (0.2 g,24%) from 5.6 M dimethylamine in ethanol (10 mL) and(6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-ol 17 (0.35 g, 0.7mmol). ¹H NMR (400 MHz, CDCl₃) δ 5.40-5.28 (m, 6H), 4.97-4.88 (m, 1H),2.35-2.24 (m, 4H), 2.24-2.19 (m, 6H), 2.08-1.93 (m, 12H), 1.70-1.55 (m,3H), 1.55-1.45 (m, 5H), 1.45-1.13 (m, 25H), 0.93-0.82 (m, 9H). Rf 0.4(10% MeOH—CH₂Cl₂).

Synthesis of Compound 20

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 6-bromohexanoate 10,(6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-yl 6-bromohexanoate 20was obtained as a colorless oil (1.4 g, 99%) from(6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-ol 17 (0.35 g, 0.7mmol), 6-Bromo-n-caproic acid (0.70 g, 3.4 mmol), EDCI (0.60 g, 3.4mmol), diisopropylethylamine (0.90 g, 6.7 mmol) and DMAP (5 mg,catalyst). Rf 0.6 (10% EtOAc-Hexanes).

Synthesis of Compound 21

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl6-(dimethylamino)hexanoate 11,(6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-yl6-(dimethylamino)hexanoate 21 was obtained as a colorless oil (1.2 g,92%) from 5.6 M dimethylamine in ethanol (15 mL). ¹H NMR (400 MHz,CDCl₃) δ 5.44-5.28 (m, 6H), 4.95-4.88 (m, 1H), 2.33-2.19 (m, 10H),2.08-1.90 (m, 12H), 1.70-1.23 (m, 9H), 1.23-1.14 (m, 26H), 0.93-0.85 (m,9H). Rf 0.15 (10% MeOH—CH₂Cl₂).

Synthetic Scheme for Compound 22

Synthesis of Compound 22

To a solution of (6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-ol 17(0.5 g, 1.1 mmol), 4-(dimethylamino)butanoic acid hydrochloride (0.3 g,1.7 mmol), EDCI hydrochloride (0.3 g, 1.7 mmol), DIPEA (0.4 g, 3.4 mmol)in anhydrous dichloromethane (10 mL) was added DMAP (5 mg). The solutionwas stirred at room temperature overnight under a nitrogen atmosphere.The mixture is concentrated in vacuo to dryness then taken up in DCM(150 mL) and extracted with saturated sodium bicarbonate. The reactionmixture was purified by column chromatography on silica gel 60 (1:1ethyl acetate/hexanes) to afford(6Z,15Z)-11-((Z)-dec-4-enyl)henicosa-6,15-dien-10-yl4-(dimethylamino)butanoate 22 as a colorless oil (0.4 g, 67%). ¹H NMR(400 MHz, CDCl₃) δ 5.40-5.28 (m, 6H), 4.97-4.90 (m, 1H), 2.36-2.25 (m,4H), 2.25-2.19 (m, 6H), 2.07-1.95 (m, 12H), 1.85-1.73 (m, 2H), 1.58-1.45(m, 3H), 1.45-1.10 (m, 24H), 0.93-0.85 (m, 9H). Rf 0.4 (10%MeOH—CH₂Cl₂).

Synthetic Scheme for Compounds 23, 24 and 25

Synthesis of Compound 23

A solution of (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 8 (0.5g, 1.1 mmol) in anhydrous diethyl ether (10 mL) was added slowly asolution of diphosgene (0.2 mL, 1.8 mmol) in anhydrous diethyl ethercooled to approximately −15° C. The solution was stirred for 1 hour thenN,N,N′-trimethyl-1,3-propanediamine (1.3 mL, 8.7 mmol) was added at −15°C. The solution was warmed to room temperature, stirred for 1 hour, andthen filtered to remove the ammonium salts and urea. The diethyl etherfiltrate was concentrated in vacuo to dryness. The residue was purifiedby column chromatography (100% ethyl acetate) to afford(6Z,16Z)-12((Z)-dec-4-enyl)docosa-6,16-dien-11-yl3-(dimethylamino)propyl(methyl)carbamate 23 as a colorless oil (0.15 g,23%), ¹H NMR (400 MHz, CDCl₃) δ 5.41-5.29 (m, 6H), 4.85-4.77 (m, 1H),3.35-3.21 (m, 2H), 2.93-2.81 (m, 3H), 2.31-2.17 (m, 8H), 2.08-1.92 (m,12H), 1.75-1.64 (m, 2H), 1.64-1.15 (m, 31H), 0.92-0.85 (m, 9H). Rf 0.45(10% MeOH—CH₂Cl₂).

Synthesis of Compound 24

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl3-(dimethylamino)propyl(methyl)carbamate 23,(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl3-(dimethylamino)propylcarbamate 24 as obtained as a colorless oil (0.1g, 17%) from (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 8 (0.5g, 1.1 mmol), diphosgene (0.2 mL, 1.8 mmol), pyridine, and3-(Dimethylamino)-1-propylamine (0.9 g, 8.7 mmol). ¹H NMR (400 MHz,CDCl₃) δ 5.44-5.28 (m, 6H), 4.81-4.72 (bs, 1H), 4.55-4.45 (bs, 1H),3.34-3.15 (m, 3H), 2.45-2.13 (m, 7H), 2.10-1.86 (m, 12H), 1.75-1.57 (m,3H), 1.57-1.03 (m, 30H), 0.93-0.85 (m, 9H). Rf 0.2 (10% MeOH—CH₂Cl₂).

Synthesis of Compound 25

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl3-(dimethylamino)propyl(methyl)carbamate 23,(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl2-(dimethylamino)ethylcarbamate 25 was obtained as a colorless oil (0.20g, 33%) from (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 8 (0.5g, 1.1 mmol), diphosgene (0.2 mL, 1.8 mmol) andN,N-dimethylethylenediamine (0.8 g, 8.7 mmol). ¹H NMR (400 MHz, CDCl₃) δ5.40-5.28 (m, 6H), 5.08-5.01 (bs, 1H), 4.82-4.73 (bs, 1H), 3.30-3.18 (m,2H), 2.44-2.35 (m, 2H), 2.30-2.20 (m, 6H), 2.07-1.91 (m, 12H), 1.65-1.11(m, 31H), 0.93-0.85 (m, 9H). Rf 0.4 (10% MeOH-DCM).

Synthetic Scheme for Compounds 26, 27 and 28

Synthesis of Compound 26

A solution of (6Z,15Z)-11-((Z)-dec-4-en-1-yl)docosa-6,15-dien-10-ol 17(2.25 g, 5.036 mmol) and pyridine (611 μL, 7.6 mmol) in anhydrous Et₂O(15 mL) was added to a cooled (0° C.) solution of diphosgene (910 μL,7.6 mmol) in Et₂O (15 mL). After stirring (10 min) the reaction mixturewas filtered and concentrated to remove the solvent and remainingphosgene gas. One-third of this chloroformate (0.879 g, 1.679 mmol) wastaken up in Et₂O (5 mL) and added to a cooled (0° C.) solution of N,Ndimethylehtylenediamine (367 μL, 3.4 mmol) in anhydrous Et₂O (5 mL).After stirring (20 min), the mixture was filtered, concentrated andsubjected to chromatography (100% EtOAc) to yield(6Z,15Z)-11-((Z)-dec-4-en-1-yl)docosa-6,15-dien-10-yl(2-(dimethylamino)ethyl)carbamate 26 (603 mg, 64%) as a clear, colorlessoil. Rf 0.28 (10% MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃, δ_(H)) 5.45-5.36(m, 6H), 5.30 (br s, 1H), 4.89-4.78 (m, 1H), 3.32-3.21 (m, 2H), 2.42 (t,2H), 2.25 (s, 6H), 2.16-1.94 (m, 12H), 1.63-1.19 (m, 29H), 0.92 (t, 9H).

Synthesis of Compound 27

A cooled (0° C.) solution of the chloroformate (0.88 g, 1.7 mmol) (asprepared in the synthesis of(6Z,15Z)-11-((Z)-dec-4-en-1-yl)docosa-6,15-dien-10-yl(2-(dimethylamino)ethyl)carbamate 26) was dissolved in anhydrous Et₂O (5mL) and added to a solution of N,N dimethylpropyldiamine (422 μL, 3.4mmol) in anhydrous Et₂O (5 mL). Upon completion (20 min), the solutionwas filtered, concentrated and then the crude material was purified bycolumn chromatography (100% EtOAc) to yield(6Z,15Z)-11-((Z)-dec-4-en-1-yl)docosa-6,15-dien-10-yl(3-(dimethylamino)propyl)carbamate 27 (675 mg, 70%) as a clear,colorless oil. Rf 0.32 (10% MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃, δ_(H))5.44-5.33 (m, 6H), 4.86-4.78 (m, 1H), 3.32-3.21 (m, 2H), 2.36 (t, 2H),2.24 (s, 6H), 2.14-1.97 (m, 12H), 1.69 (app. p, 2H), 1.61-1.50 (m, 3H),1.50-1.20 (m, 27H), 0.91 (t, 9H).

Synthesis of Compound 28

A cooled (0° C.) solution of the chloroformate (0.88 g, 1.7 mmol) (asprepared in the synthesis of(6Z,15Z)-11-((Z)-dec-4-en-1-yl)docosa-6,15-dien-10-yl(2-(dimethylamino)ethyl)carbamate 26) was dissolved in anhydrous Et₂O (5mL) and added to a solution of N,N,N′ trimethylpropyldiamine (492 μL,3.4 mmol) in anhydrous Et₂O (5 mL). Upon completion (20 min), thesolution was filtered, concentrated and then the crude material waspurified by column chromatography (100% EtOAc) to yield(6Z,15Z)-11-((Z)-dec-4-en-1-yl)henicosa-6,15-dien-10-yl(3-(dimethylamino)propyl)(methyl) carbamate 28 (672 mg, 68%) as a clear,colorless oil. Rf 0.44 (10% MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃, δ_(H))5.43-5.32 (m, 6H), 4.85 (br. s, 1H), 3.38-3.27 (m, 2H), 2.95-2.87 (m,3H), 2.28 (t, 2H), 2.24 (s, 6H), 2.14-1.96 (m, 12H), 1.72 (app. p, 2H),1.67-1.49 (m, 3H), 1.49-1.20 (m, 26H), 0.91 (t, 9H).

Synthetic Scheme for Compounds 30 and 31

Synthesis of Compound 29

A 100 mL round bottom flask was charged with magnesium turnings (263 mg,10.9 mmol) and a stirbar. The flask was dried with a heat gun for 5minutes, cooled under nitrogen before THF (5 mL) and a small grain ofiodine was added. A solution of (Z)-1-bromodec-4-ene 3 (2 g, 9.1 mmol)in THF (5 mL) was added slowly. The solution was stirred at roomtemperature for 2 hours then ethyl glyoxalate (0.375 mL, 1.82 mmol, 50%solution in toluene) was added. Upon completion, the solution wasquenched with saturated ammonium chloride solution (5 mL) and stirreduntil the excess magnesium had dissolved. The solution was diluted withwater and extracted with ethyl acetate (3×50 mL). The combined extractswere dried on magnesium sulfate, filtered and concentrated in vacuo todryness. The residue was purified by column chromatography (100% Hexanesto 20% EtOAc in hexanes) to afford(6Z,16Z)-11-((Z)-dec-4-enyl)docosa-6,16-diene-11,12-diol 29 as acolorless oil (700 mg, 48%).

Synthesis of Compound 30

Using an analogous procedure to that described for the synthesis of6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl4-(dimethylamino)butanoate 9,(6Z,16Z)-12-((Z)-dec-4-enyl)-12-hydroxydocosa-6,16-dien-11-yl4-(dimethylamino)butanoate 30 was obtained as a colorless oil (0.10 g,25%) from (6Z,16Z)-11-((Z)-dec-4-enyl)docosa-6,16-diene-11,12-diol (0.35g, 0.7 mmol), 4-(dimethylamino)butanoic acid hydrochloride (0.20 g, 1.1mmol), EDCI (0.20 g, 1.1 mmol), diisopropylethylamine (0.3 g, 2.2 mmol)and DMAP (5 mg, catalyst). ¹H NMR (400 MHz, CDCl₃) δ 5.44-5.28 (m, 6H),5.03-4.95 (m, 11-1), 2.25-2.17 (m, 6H), 2.14-1.65 (m, 14H), 1.65-1.10(m, 33H), 0.93-0.82 (m, 9H). Rf 0.2 (10% MeOH—CH₂Cl₂).

Synthesis of Compound 31

Using an analogous procedure to that described for the synthesis of6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl4-(dimethylamino)butanoate 9,(6Z,16Z)-12-((Z)-dec-4-enyl)-12-hydroxydocosa-6,16-dien-11-yl3-(dimethylamino)propanoate 31 was obtained as a colorless oil (0.4 g,33%) from (6Z,16Z)-11-((Z)-dec-4-enyl)docosa-6,16-diene-11,12-diol (1.0g, 2.1 mmol), 4-(dimethylamino)butanoic acid hydrochloride (0.50 g, 3.1mmol), EDCI (0.60 g, 3.1 mmol), diisopropylethylamine (0.80 g, 6.3 mmol)and DMAP (5 mg, catalyst). ¹H NMR (400 MHz, CDCl₃) δ 5.40-5.28 (m, 6H),5.12-5.07 (m, 1H), 4.75-4.55 (bs, 2.77-2.65 (m, 1H), 2.65-2.41 (m, 3H),2.28-2.15 (m, 6H), 2.15-1.92 (m, 12H), 1.67-1.10 (m, 30H), 0.93-0.82 (m,9H). Rf 0.5 (10% MeOH—CH₂Cl₂).

Synthetic Scheme for Compound 40

Synthesis of Compound 33

Using an analogous procedure to that described for the synthesis of 2,(Z)-non-3-enyl methanesulfonate 33 was obtained as a yellow oil (33 g,85%) from (Z)-non-3-en-1-ol 32 (25.0 g, 176 mmol), triethylamine (25.0mL) and methane sulfonyl chloride (27.2 mL, 352 mmol). Rf 0.68 (CH₂Cl₂).

Synthesis of Compound 34

Using an analogous procedure to that described for the synthesis of 3,(Z)-non-3-enyl bromide 34 was obtained as a yellow oil (20.2 g, 85%)from (Z)-non-3-enyl methanesulfonate (25.7 g, 117 mmol) andtetrabutylammonium bromide (52.6 g, 163 mmol). Rf 0.73 (hexanes).

Synthesis of Compound 35

Using an analogous procedure to that described for the synthesis of 4,(6Z,13Z)-nonadeca-6,13-dien-10-ol 35 (9.11 g, 85%) was obtained as acolorless oil from (Z)-non-3-enyl bromide (15.8 g, 76.8 mmol), magnesiumturnings (2.0 g, 82 mmol), ethyl formate (6.36 mL, 79.1 mmol) andpotassium hydroxide (3.88 g, 69.1 mmol). Rf 0.43 (10% EtOAc-hexanes).

Synthesis of Compound 36

Using an analogous procedure to that described for the synthesis of 5,(6Z,13Z)-nonadeca-6,13-dien-10-yl methanesulfonate 36 (11.6 g, 99%) wasobtained as a colorless oil from (6Z,13Z)-nonadeca-6,13-dien-10-ol (9.11g, 32.5 mmol), triethylamine (10 mL) and methane sulfonyl chloride (5.0mL, 65 mmol). Rf 0.73 (CH₂Cl₂).

Synthesis of Compound 37

Using an analogous procedure to that described for the synthesis of 6,(Z)-2-((Z)-non-3-en-1-yl)undec-5-enenitrile 37 (7.2 g, 77%) was obtainedas a colorless oil from (6Z,13Z)-nonadeca-6,13-dien-10-ylmethanesulfonate (11.6 g, 32.3 mmol) and sodium cyanide (3.96 g, 80.9mmol). Rf 0.75 (10% EtOAc-hexanes).

Synthesis of Compound 38

Using an analogous procedure to that described for the synthesis of 7,(Z)-2-((Z)-non-3-en-1-yl)undec-5-enal 38 (5.0 g, 69%) was obtained as acolorless oil from (Z)-2-((Z)-non-3-en-1-yl)undec-5-enenitrile (7.2 g,24.9 mmol) and DIBAL (49.7 mL as a 1M solution in hexanes, 49.7 mmol).Rf 0.69 (10 EtOAc-hexanes).

Synthesis of Compound 39

Using an analogous procedure to that described for the synthesis of 8,(6Z,14Z)-11-((Z)-non-3-en-1-yl)icosa-6,14-dien-10-ol 39 (1.64 g, 76%)was obtained as a colorless oil from(Z)-2-((Z)-non-3-en-1-yl)undec-5-enal (1.5 g, 5.1 mmol), (Z)-non-3-enylbromide (1.58 g, 7.7 mmol) and magnesium turnings (206 mg, 8.5 mmol). Rf0.46 (10% EtOAc-hexanes).

Synthesis of Compound 40

Using an analogous procedure to that described for the synthesis of 9,(6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl4-(dimethylamino)butanoate 40 (483 mg, 76%) was obtained as a colorlessoil from (6Z,14Z)-11-((Z)-non-3-en-1-yl)icosa-6,14-dien-10-ol (500 mg,1.19 mmol), EDC (686 mg, 3.58 mmol), Hünig's base (726 μL, 4.17 mmol)and N,N dimethylaminobutyric acid hydrochloride (600 mg, 3.58 mmol). Rf0.43 (10% CH₃OH—CH₂Cl₂).

Synthetic Scheme for Compound 42

Synthesis of Compound 41

Using an analogous procedure to that described for the synthesis of 10,(6Z,14Z)-11-((Z)-non-3-en-1-yl)icosa-6,14-dien-10-yl 5-bromopentanoate41 (655 mg, 95%) was obtained as a colorless oil from(6Z,14Z)-11-((Z)-non-3-en-1-yl)icosa-6,14-dien-10-ol (500 mg, 1.19mmol), EDC (686 mg, 3.58 mmol) and 5-bromovaleric acid (649 mg, 3.58mmol). Rf 0.54 (5% EtOAc-hexanes).

Synthesis of Compound 42

Using an analogous procedure to that described for the synthesis of 11,(6Z,14Z)-11-((Z)-non-3-en-1-yl)icosa-6,14-dien-10-yl5-(dimethylamino)pentanoate 42 (421 mg, 68%) was obtained as a colorlessoil from (6Z,14Z)-11-((Z)-non-3-en-1-yl)icosa-6,14-dien-10-yl5-bromopentanoate (655 mg, 1.13 mmol) and dimethylamine (25 mL as a 5.6Msolution in EtOH). Rf 0.4 (10% CH₃OH—CH₂Cl₂).

Synthetic Scheme for Compound 50

Synthesis of Compound 44

Using an analogous procedure to that described for the synthesis of 4,henicosan-11-ol 44 (7.06 g, 99%) was obtained as a colorless oil frombromodecane (9.4 mL, 45.2 mmol), magnesium turinings (1.18 g, 48.4mmol), ethyl formate (3.74 mL, 46.6 mmol) and potassium hydroxide (2.28g, 40.7 mmol). Rf 0.36 (10% EtOAc-hexanes), FW 312.57, C₂₁H₄₄O.

Synthesis of Compound 45

Using an analogous procedure to that described for the synthesis of 5,henicosan-11-yl methanesulfonate 45 (6.87 g, 78%) was obtained as acolorless oil from henicosan-11-ol (7.06 g, 22.6 mmol), triethylamine(22 mL) and methane sulfonyl chloride (3.5 mL, 45 mmol). Rf 0.86(CH₂Cl₂).

Synthesis of Compound 46

Using an analogous procedure to that described for the synthesis of 6,2-decyldodecanenitrile 46 (2.25 g, 40%) was obtained as a colorless oilfrom henicosan-11-yl methanesulfonate (6.87 g, 17.6 mmol) and sodiumcyanide (4.31 g, 87.9 mmol). Rf 0.84 (10% EtOAc-hexanes).

Synthesis of Compound 47

Using an analogous procedure to that described for the synthesis of 7,2-decyldodecanal 47 (1.91 g, 84%) was obtained as a colorless oil from2-decyldodecanenitrile (2.25 g, 7.0 mmol) and DIBAL (14 mL, as a 1Msolution in hexanes, 14 mmol). Rf 0.51 (5% EtOAc-hexanes).

Synthesis of Compound 48

Using an analogous procedure to that described for the synthesis of 8,(Z)-12-decyldocos-6-en-11-ol 48 (1.08 g, 40%) was obtained as acolorless oil from 2-decyldodecanal (1.91 g, 5.87 mmol), (Z)-dec-4-enylbromide (1.45 g, 7.05 mmol) and magnesium turnings (183 mg, 7.54 mmol).Rf 0.26 (10% EtOAc-hexanes).

Synthesis of Compound 49

Using an analogous procedure to that described for the synthesis of 10,(Z)-12-decyldocos-6-en-11-yl 5-bromopentanoate 49 (916 mg, 63%) wasobtained as a colorless oil from (Z)-12-decyldocos-6-en-11-01 (1.08 g,2.33 mmol), EDC (804 mg, 4.19 mmol) and 5-bromovaleric acid (1.27 g,6.99 mmol). Rf 0.29 (5% EtOAc-hexanes).

Synthesis of Compound 50

Using an analogous procedure to that described for the synthesis of 11,(Z)-12-decyldocos-6-en-11-yl 5-(dimethylamino)pentanoate 50 (662 mg,80%) was obtained as a colorless oil from (Z)-12-decyldocos-6-en-11-yl5-bromopentanoate (916 mg, 1.16 mmol) and dimethylamine (27 mL as a 5.6Msolution in EtOH). Rf 0.51 (10% CH₃OH—CH₂Cl₂).

Synthetic Scheme for Compound 53

Synthesis of Compound 51

Using an analogous procedure to that described for the synthesis of 8,(Z)-12-((Z)-dec-4-en-1-yl)docos-16-en-11-ol 51 (3.37 g, 65%) wasobtained as a colorless oil from (Z)-2-((Z)-dec-4-enyl)dodec-6-enal 7(3.6 g, 11.2 mmol), 1-bromodecane (3.5 mL, 16.9 mmol) and magnesiumturnings (438 mg, 18.0 mmol). Rf 0.31 (5% EtOAc-hexanes).

Synthesis of Compound 52

Using an analogous procedure to that described for the synthesis of 10,(Z)-12-((Z)-dec-4-en-1-yl)docos-16-en-11-yl 5-bromopentanoate 52 (4.69g, 99%) was obtained as a colorless oil from(Z)-12-((Z)-dec-4-en-1-yl)docos-16-en-11-ol (3.37 g, 7.29 mmol), EDC(2.51 g, 13.1 mmol) and 5-bromovaleric acid (3.96 g, 21.8 mmol). Rf 0.56(5% EtOAc-hexanes).

Synthesis of Compound 53

Using an analogous procedure to that described for the synthesis of 11,(Z)-12-decyldocos-6-en-11-yl 5-(dimethylamino)pentanoate 53 (943 mg,99%) was obtained as a colorless oil from (Z)-12-decyldocos-6-en-11-yl5-bromopentanoate (1.0 g, 1.6 mmol) and dimethylamine (30 mL as a 5.6Msolution in EtOH). Rf 0.50 (10% CH₃OH—CH₂Cl₂).

Example 2

This Example compares the effectiveness, in a murine ApoB siRNA activitymodel, of short chain trialkyl lipids of the present invention withlipids having longer alkyl chains but which are otherwise structurallyidentical to the short chain trialkyl lipids.

Nucleic acid-lipid particle formulations containing cationic lipids wereassessed for their ability to knockdown ApoB expression in the livers of7 to 9 week old female BALB/c mice. Mice were dosed intravenously (tailvein) in groups of three at either 0.02, 0.03 or 0.05 mg/kg. ApoBknockdown (normalized to the housekeeping gene GAPDH) was measuredrelative to PBS as a negative control. Each experiment was terminated at48 hours after dosing.

For comparison to a positive control, the performance of short chaintrialkyl lipids of the present invention was compared to a potentcationic lipid, referred to as C2K, that is known to facilitate nucleicacid delivery, in vivo, in nucleic acid-lipid particles (NatureBiotech., Vol 28(2), 172 (2010)). C2K has the following structure:

As shown in Table 1, at an injected dose of 0.02 mg/kg, 10 out of 11cationic lipids of the present invention (compounds 9, 13, 14, 19, 22,27, 40, 42, 50 and 53) displayed greater activity than C2K.

TABLE 1 ApoB silencing (0.02 mg/kg siRNA) for various trialkyl cationiclipids of the present invention. ApoB gene silencing relative to PBScontrol (Liver ApoB:GAPD siRNA Dose Compound mRNA ratio) 0.02 mg/kg C2K−61% 9 −73% 13 −80% 14 −69% 19 −79% 22 −79% 24 −48% 27 −68% 40 −80% 42−77% 50 −72% 53 −78%

As shown in Table 2, in a separate experiment, at an injected dose of0.03 mg/kg, 5 out of 7 cationic lipids of the present invention(compounds 11, 13, 19, 23, and 25) displayed greater activity than C2K.

TABLE 2 ApoB silencing (0.03 mg/kg siRNA) for various short chaintrialkyl cationic lipids ApoB gene silencing relative to PBS control(Liver ApoB:GAPD siRNA Dose Compound mRNA ratio) 0.03 mg/kg C2K −54% 9−49% 11 −78% 13 −84% 19 −87% 23 −80% 25 −63% 30 −30%

The activity of cationic lipids of the present invention was alsocompared to corresponding trialkyl cationic lipids that are structurallyidentical to the lipids of the present invention, except that the alkylchains are longer. The structures of these longer chain trialkylcationic lipids (identified as compounds 54, 55, 56, 57, 58, 59 and 60)are shown in Table 3.

TABLE 3 structures of long chain trialkyl cationic lipids

54

55

56

57

58

59

60

Compounds 54, 55, 56, 57, 58, 59 and 60 were prepared according to theprocedures described in U.S. patent application Ser. No. 13/235,253,filed on Sep. 16, 2011, which is incorporated herein by reference in itsentirety.

As shown in Table 4, the longer chain lipids 54, 55, 56, 57, 58, 59, 60were dosed at 0.05 mg/kg (2.5 times the dose described in Table 1), anddisplayed ApoB knockdown ranging from +25% to −69% compared to 60% forC2K (i.e., some compounds displayed a moderate improvement over C2K).

TABLE 4 ApoB silencing (0.05 mg/kg siRNA) for various trilinoleylcationic lipids ApoB gene silencing relative to PBS control (LiverApoB:GAPD siRNA Dose Compound mRNA ratio) 0.05 mg/kg C2K  −60%^(a) 54 −6% 55 −28% 56 −23% 57 +25% 58 −62% 59 −69% 60 −63% ^(a)Average ApoBsilencing over four studies

In general, the activity of the shorter chain (C9 to C10) trialkylcationic lipids of the present invention was substantially improved whencompared to the corresponding longer chain, trilinoleyl (C18),counterpart lipid. For example, a direct comparison of compound 13 withits trilinoleyl variant 54 showed an improvement from −6% (0.05 mg/kg)to −80% (0.02 mg/kg) in ApoB knockdown, despite the fact that compound13 was dosed 2.5 times lower. The same trend was observed when comparingcompounds 55 to 11, 56 to 24 and 57 to 25.

Example 3

Further experiments in the murine ApoB model used a different cationiclipid as the positive control; DLin-MP-DMA. DLin-MP-DMA is described inpatent application WO 2011/141705, and has the structure:

As shown in Table 5, in the same murine ApoB model, DLin-MP-DMA wasshown to be more effective than C2K in three separate experiments, andis therefore a valid positive control:

TABLE 5 Comparison between C2K and Dlin-MP-DMA as Positive Controls ApoBgene silencing relative to PBS control (Liver ApoB:GAPD siRNA DoseCompound mRNA ratio) Experiment 1 0.03 mg/kg C2K −51% DLin-MP-DMA −69%Experiment 2 0.05 mg/kg C2K −59% DLin-MP-DMA −65% Experiment 3 0.03mg/kg C2K −54% DLin-MP-DMA −62%

In another experiment, two more lipids of the present invention(Compounds 62 and 71) were formulated into lipid nanoparticles withsiRNA to target ApoB. Mice were dosed intravenously (tail vein) andsacrificed 48 h after dosing. Livers were harvested and homogenized, andthe level of ApoB silencing (normalized to the housekeeping gene GAPDH)then measured via Quantigene Assay. The results are shown in Table 6 andare expressed as a percentage, relative to a PBS-treated negativecontrol group. The siRNA sequence used in this experiment was different,and less potent than that used in Example 2. Inter-experimentcomparisons are therefore not possible:

TABLE 6 ApoB silencing for various trialkyl cationic lipids of thepresent invention. ApoB gene silencing relative to PBS control (LiverApoB:GAPD siRNA Dose Compound mRNA ratio) 0.04 mg/kg DLin-MP-DMA −32%Compound 62 −56% Compound 71 −29%

Compound 71 had similar activity to the DLin-MP-DMA control. Compound 62was significantly more active. The synthesis of these and othercompounds is described in Example 4.

Example 4

This Examples describes the synthesis of additional compounds of thepresent invention.

Synthetic Scheme for Compound 62

Synthesis of Compound 61

Using an analogous procedure to that described for the synthesis of 5,(6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl methanesulfonate61 (1.18 g, 90%) was obtained as a colorless oil from(6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-ol 8 (1.12 g, 2.43mmol), triethylamine (8 mL) and methane sulfonyl chloride (0.38 mL, 4.9mmol). Rf 0.91 (CH₂Cl₂).

Synthesis of Compound 62

A solution of the mesylate 61 (1.09 g, 2.01 mmol) in toluene (30 mL) wassuccessively treated with N,N-dimethylaminobutanol (1.34 mL, 10.1 mmol)and NaH (442 mg as a 60% dispersion in oil, 11.1 mmol). Once gasevolution ceased the reaction mixture was brought to reflux (115° C.bath temp.) and stirred (50 h). The reaction mixture was then cooled(rt) and poured into cold water and then extracted with EtOAc. Thecombined organics were washed with water and brine, dried (Na₂SO₄),filtered, concentrated and purified via chromatography (100% EtOAc) toyield4-(((6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl)oxy)-N,N-dimethylbutan-1-amine62 (143 mg, 13%) as a pale yellow oil. ¹H NMR (400 MHz, CDCl₃) δ5.41-5.30 (m, 6H), 3.46-3.35 (m, 2H), 3.19-3.14 (m, 1H), 2.34 (t, 2H),2.24 (s, 6H), 2.10-1.93 (m, 12H), 1.60-1.09 (m, 35H), 0.90 (t, 9H). Rf0.54 (10% MeOH—CH₂Cl₂).

Synthetic Scheme for Compound 71

Synthesis of Compound 63

Using an analogous procedure to that described for the synthesis of 5,3,7-dimethyloctyl methanesulfonate 63 (7.47 g, >99%) was obtained as acolorless oil from 3,7-dimethyloctan-1-ol (5.0 g, 31.6 mmol),triethylamine (8 mL) and methane sulfonyl chloride (4.89 mL, 63.2 mmol).Rf 0.69 (CH₂Cl₂).

Synthesis of Compound 64

Using an analogous procedure to that described for the synthesis of 3,1-bromo-3,7-dimethyloctane 64 was obtained as a colorless oil (6.0 g,86%) from 3,7-dimethyloctyl methanesulfonate 63 (7.47 g, 31.6 mmol) andtetrabutylammonium bromide (13.2 g, 41.1 mmol). Rf 0.92 (Hexanes).

Synthesis of Compound 65

Using an analogous procedure to that described for the synthesis of 4,2,6,12,16-tetramethylheptadecan-9-ol 65 (7.0 g, quantitative) wasobtained as a colorless oil from 1-bromo-3,7-dimethyloctane 64 (10 g,45.2 mmol), magnesium turnings (1.21 g, 49.8 mmol), ethyl formate (3.8mL, 47.5 mmol) and potassium hydroxide (3.8 g, 67.8 mmol). Rf 0.38 (10%EtOAc-hexanes).

Synthesis of Compound 66

Using an analogous procedure to that described for the synthesis of 5,2,6,12,16-tetramethylheptadecan-9-yl methanesulfonate 66 (1.56 g, 87%)was obtained as a colorless oil from2,6,12,16-tetramethylheptadecan-9-ol 65 (1.39 g, 5.14 mmol),triethylamine (3 mL) and methane sulfonyl chloride (0.8 mL, 10.3 mmol).Rf 0.8 (CH₂Cl₂).

Synthesis of Compound 67

Using an analogous procedure to that described for the synthesis of 6,2-(3,7-dimethyloctyl)-5,9-dimethyldecanenitrile 67 (0.8 g, 56%) wasobtained as a colorless oil from 2,6,12,16-tetramethylheptadecan-9-ylmethanesulfonate 66 (1.56 g, 4.48 mmol) and sodium cyanide (0.55 g, 11.2mmol). Rf 0.8 (10% EtOAc-hexanes).

Synthesis of Compound 68

Using an analogous procedure to that described for the synthesis of 7,2-(3,7-dimethyloctyl)-5,9-dimethyldecanal 68 (0.63 g, 78%) was obtainedas a colorless oil from 2-(3,7-dimethyloctyl)-5,9-dimethyldecanenitrile67 (0.8 g, 2.49 mmol) and DIBAL (5.74 mL as a 1M solution in hexanes,5.74 mmol). Rf 0.6 (10% EtOAc-hexanes).

Synthesis of Compound 69

Using an analogous procedure to that described for the synthesis of 8,10-(3,7-dimethyloctyl)-2,6,13,17-tetramethyloctadecan-9-ol 69 (0.53 g,62%) was obtained as a colorless oil from2-(3,7-dimethyloctyl)-5,9-dimethyldecanal 68 (0.6 g, 1.85 mmol),1-bromo-3,7-dimethyloctane 64 (2.0 g, 9.0 mmol) and magnesium turnings(232 mg, 9.67 mmol). Rf 0.37 (10% EtOAc-hexanes).

Synthesis of Compound 70

Using an analogous procedure to that described for the synthesis of 10,1043,7-dimethyloctyl)-2,6,13,17-tetramethyloctadecan-9-yl5-bromopentanoate 68 (450 mg, crude) was obtained as a yellow oil from10-(3,7-dimethyloctyl)-2,6,13,17-tetramethyloctadecan-9-ol 69 (200 mg,0.43 mmol), EDC (246 mg, 1.28 mmol) and 5-bromovaleric acid (246 mg,1.28 mmol). Rf 0.49 (5% EtOAc-hexanes).

Synthesis of Compound 71

Using an analogous procedure to that described for the synthesis of 11,1043,7-dimethyloctyl)-2,6,13,17-tetramethyloctadecan-9-yl5-(dimethylamino)pentanoate 71 (184 mg, 72% 2 steps) was obtained as acolorless oil from10-(3,7-dimethyloctyl)-2,6,13,17-tetramethyloctadecan-9-yl5-bromopentanoate 68 (450 mg crude) and dimethylamine (10 mL as a 2.0Msolution in EtOH). ¹H NMR (400 MHz, CDCl₃) δ 4.95-4.87 (m, 1H), 2.33 (t,2H), 2.28 (t, 2H), 2.23 (s, 6H), 1.74-1.60 (m, 4H), 1.58-0.99 (m, 37H),0.93-0.79 (m, 27H). Rf 0.43 (10% CH₃OH—CH₂Cl₂).

Synthetic Scheme for Compound 74

Synthesis of Compound 72

Using an analogous procedure to that described for the synthesis of 8,(Z)-10-((Z)-dec-4-en-1-yl)-2,6-dimethylicos-14-en-9-ol 72 (0.62 g, 72%)was obtained as a colorless oil from (Z)-2-((Z)-dec-4-enyl)dodec-6-enal7 (0.6 g, 1.87 mmol), 1-bromo-3,7-dimethyloctane 64 (3.9 g, 17.5 mmol)and magnesium turnings (454 mg, 18.7 mmol). Rf 0.61 (10% EtOAc-hexanes).

Synthesis of Compound 73

Using an analogous procedure to that described for the synthesis of 10,(Z)-10-((Z)-dec-4-en-1-yl)-2,6-dimethylicos-14-en-9-yl 5-bromopentanoate73 (900 mg, crude) was obtained as a yellow oil from(Z)-10-((Z)-dec-4-en-1-yl)-2,6-dimethylicos-14-en-9-ol 72 (620 mg, 1.34mmol), EDC (500 mg, 2.6 mmol) and 5-bromovaleric acid (500 mg, 2.56mmol). Rf 0.72 (10% EtOAc-hexanes).

Synthesis of Compound 74

Using an analogous procedure to that described for the synthesis of 11,(Z)-10-((Z)-dec-4-en-1-yl)-2,6-dimethylicos-14-en-9-yl5-(dimethylamino)pentanoate 74 (466 mg, 58% 2 steps) was obtained as acolorless oil from(Z)-10-((Z)-dec-4-en-1-yl)-2,6-dimethylicos-14-en-9-yl 5-bromopentanoate73 (900 mg, crude) and dimethylamine (15 mL as a 2.0M solution in EtOH).¹H NMR (400 MHz, CDCl₃) δ 5.43-5.29 (m, 4H), 4.94-4.88 (m, 1H), 2.32 (t,2H), 2.26 (t, 2H), 2.15 (s, 6H), 2.08-1.93 (m, 8H), 1.70-1.00 (m, 43H),0.95-0.83 (m, 15H). Rf 0.42 (10% CH₃OH—CH₂Cl₂).

Synthetic Scheme for Compound 76

Synthesis of Compound 75

A solution of 5-bromopentan-1-ol (1.0 g, 5.99 mmol) was prepared withdimethylamine (10 mL, as a 2M solution in EtOH) in a sealed vessel andheated (80° C.). After stirring (16 h) the dimethylamine and EtOH wereremoved under reduced pressure to yield 5-(dimethylamino)pentan-1-olhydrobromide 75 (1.26 g, quantitative) as a yellow-orange solid. Rf 0.25(10% CH₃OH—CH₂Cl₂).

Synthesis of Compound 76

Using an analogous procedure to that described for the synthesis of 62,5-(((6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl)oxy)-N,N-dimethylpentan-1-amine76 (864 mg, 37%) was obtained as a pale yellow oil from(6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl methanesulfonate61 (1.86 g, 3.45 mmol), 5-(dimethylamino)pentan-1-ol hydrobromide 75(1.26 g, 5.99 mmol) and NaH (288 mg as a 60% dispersion in oil, 7.2mmol). ¹H NMR (400 MHz, CDCl₃) δ 5.43-5.32 (m, 6H), 3.46-3.33 (m, 2H),3.18-3.12 (m, 1H), 2.30-2.18 (m, 8H), 2.07-1.93 (m, 12H), 1.61-1.04 (m,37H), 0.89 (t, 9H). Rf 0.47 (10% MeOH—CH₂Cl₂).

Synthetic Scheme for Compound 79

Synthesis of Compound 77

To a cooled solution (−15° C.) of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 8 (5 g, 10.9 mmol) inanhydrous dichloromethane (125 mL) under nitrogen was added, drop-wise,diethyl zinc (1M in hexane, 82 mL, 81.8 mmol) over 20 minutes. Thesolution was stirred for 70 minutes at 0° C. then diiodomethane (6.6 mL,81.8 mmol) was carefully added. The solution was stirred overnight,allowing it to warm to room temperature. Upon completion, the solutionwas poured into ice water (350 mL) and diluted with ethyl acetate (450mL). Then 5% HCl (350 mL) was added to help alleviate the emulsion thathad formed. The organic layer was washed with NaHCO₃ (sat. aq. 500 mL),water (500 mL) and brine (500 mL). The combined aqueous layers were backextracted with ethyl acetate. The combined organic extracts were driedon magnesium sulphate, filtered and concentrated in vacuo to dryness.The residue was purified by column chromatography on silica gel (2.5%ethyl acetate in hexanes) to afford a pink colored oil. To remove thecolor (IA the purified product was dissolved in dichloromethane (150 mL)and washed with Na₂S₂O₃ (sat. aq. 2×40 mL) to afford1,8-bis(2-pentylcyclopropyl)-5-(3-(2-pentylcyclopropyl)propyl)octan-4-ol77 as a pale yellow oil (5.54 g, 96.5%).

Synthesis of Compound 78

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 6 bromohexanoate 10,1,8-bis(2-pentylcyclopropyl)-5-(3-(2-pentylcyclopropyl)propyl)octan-4-yl6-bromohexanoate was obtained as a crude oil from1,8-bis(2-pentylcyclopropyl)-5-(3-(2-pentylcyclopropyl)propyl)octan-4-yl6-(dimethylamino)hexanoate (0.75 g, 1.5 mmol), anhydrous dichloromethane(5.2 ml), 6-bromohexanoic acid (0.88 g, 4.5 mmol),1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.87 g, 4.5mmol), and 4-dimethylaminopyridine (5 mg). The product was used in thenext step without further purification.

Synthesis of Compound 79

Using an analogous procedure to that describe for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl6-(dimethyamino)hexanoate 11, was obtained as an oil (0.56 g, 59%) from1,8-bis(2-pentylcyclopropyl)-5-(3-(2-pentylcyclopropyl)propyl)octan-4-yl 6-bromohexanoate (1.0 g, 1.5 mmol) and 2.0Mdimethylamine in ethanol (3.5 ml). Rf 0.50 (10% MeOH—CH₂Cl₂).

Synthetic Scheme for Compound 83

Synthesis of Compound 80

To a solution of 2-octyldodecan-1-ol (20 g, 67.0 mmol) in anhydrousdichloromethane (500 mL) was added pyridinium chlorochromate (43.2 g,200 mmol). The solution was stirred for 3 hours at room temperature thenfiltered through a pad of silica and eluted with dichloromethane toafford 2-octyldodecanal 80 as a colorless oil (10.5 g, 50%).

Synthesis of Compound 81

Using an analogous procedure to that described for the synthesis of(6Z,15Z)-henicosa-6,15-dien-11-ol 4, (Z)-12-octyldocos-6-en-11-ol 81 wasobtained as a colorless oil (0.67 g, 92%) from 2-octyldodecanal 80 (0.5g, 1.6 mmol), (Z)-1-bromodece-4-ene (0.7 g, 3.1 mmol), magnesium (80 mg,3.4 mmol), anhydrous tetrahydrofuran (0.5 mL), water (2 mL), EtOH (2 mL)and KOH (0.2 g, 2.8 mmol).

Synthesis of Compound 82

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 6 bromohexanoate 10,(Z)-12-octyldocos-6-en-11-yl 6-bromohexanoate 82 was obtained as acolorless oil (0.78 g, 85%) from (Z)-12-octyldocos-6-en-11-ol 81 (0.67g, 1.5 mmol), anhydrous dichloromethane (5 mL), 6-bromohexanoic acid(0.80 g, 4.4 mmol), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (0.85 g, 4.4 mmol) and 4-dimethylaminopyridine (5 mg). Theproduct was used in the next step without further purification.

Synthesis of Compound 83

Using an analogous procedure to that describe for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl6-(dimethyamino)hexanoate 11, (Z)-12-octyldocos-6-en-11-yl6-(dimethylamino)hexanoate 83 was obtained as an oil (103 mg, 14%) from(Z)-12-octyldocos-6-en-11-yl 6-bromohexanoate 82 (0.78 g, 1.3 mmol) and2.0M dimethylamine in ethanol (3 mL). ¹H NMR (400 MHz, CDCl₃) δ5.43-5.26 (m, 2H), 4.96-4.91 (m, 1H), 2.33 (t, 4H), 2.26 (s, 6H),2.08-1.93 (m, 4H), 1.70-1.60 (m, 21-1), 1.57-1.43 (m, 4H), 1.40-1.15 (m,43H), 0.94-0.85 (m, 9H).

Synthetic Scheme for Compound 89

Synthesis of Compound 84

To a cooled solution (0° C.) of (E)-ethyl dec-4-enoate (20 g, 101 mmol)in anhydrous diethyl ether (350 mL) was added lithium aluminum hydride(8.9 g, 212 mmol) under nitrogen. The solution was stirred for 1 hour atroom then cooled to 0° C. and quenched slowly with 5M NaOH (30 mL) anddiluted with ethyl ether (100 mL). The solution was stirred for 30 minand dried on magnesium sulfate, filtered and concentrated vacuo todryness to afford (E)-dec-4-en-1-ol 84 as oil (16.1 g, quantitative).

Synthesis of Compound 85

Using an analogous procedure to that described for the synthesis of(Z)-dec-4-enyl methanesulfonate 2, (E)-dec-4-enyl methanesulfonate 85was obtained as an orange oil (32.7 g) from (E)-dec-4-en-1-ol (16.1 g,94.7 mmol), triethylamine (15.5 mL, 111.7 mmol) and methane sulfonylchloride (15.6 mL, 201.7 mmol). Rf 0.65 (100% CH₂Cl₂).

Synthesis of Compound 86

Using an analogous procedure to that described for the synthesis of(Z)-1-bromodec-4-ene 3, (E)-1-bromodec-4-ene 86 was obtained as oil(17.9 g, 81%) from (E)-dec-4-enyl methanesulfonate 85 (23.5 g, 94.7mmol), and tetrabutylammonium bromide (40.0 g, 124.1 mmol). Rf 0.85 (10%EtOAc-Hexanes).

Synthesis of Compound 87

Using an analogous procedure to that described for the synthesis ofafford (6Z,15Z)-henicosa-6,15-dien-11-ol 4,(6E,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 87 was obtained asoil (1.28 g, 71%) from (E)-1-bromodec-4-ene 86 (1.7 g, 7.8 mmol),magnesium turnings (0.19 g, 7.8 mmol),(Z)-2-((Z)-dec-4-enyl)dodec-6-enal 7 (1.25 g, 3.9 mmol) and potassiumhydroxide (0.66 g, 11.7 mmol). Rf 0.43 (10% EtOAc-Hexanes).

Synthesis of Compound 88

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 6-bromohexanoate 10,(6E,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 5-bromopentanoate 88was obtained as oil (1.36 g, 78%) from(6E,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ol 87 (1.28 g, 2.8mmol), and 5-bromo-n-valeric acid (1.00 g, 5.6 mmol),1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.06 g, 5.6mmol), diisopropylethylamine (1.1 g, 83.0 mmol) anddimethylaminopyridine (10 mg).

Synthesis of Compound 89

Using an analogous procedure to that described for the synthesis of(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl6-(dimethylamino)hexanoate 11,(6E,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl5-(dimethylamino)pentanoate 89 was obtained as oil (0.45 g, 35%) from(6E,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 5-bromopentanoate 88(1.36 g, 2.2 mmol), and 2 M dimethylamine in ethanol (5 mL) ¹H NMR (400MHz, CDCl₃) δ 5.45-5.28 (m, 6H), 4.96-4.91 (m, 1H), 2.34-2.25 (m, 2H),2.06-1.90 (m, 12H), 1.68-1.61 (m, 2H), 1.55-1.45 (m, 5H), 1.45-1.16 (m,28H), 0.95-0.84 (m, 9H). Rf 0.46 (10% MeOH—CH₂Cl₂).

Synthetic Scheme for Compound 90

Synthesis of Compound 89

Using an analogous procedure to that described for the synthesis of 5,1,8-bis(2-pentylcyclopropyl)-5-(3-(2-pentylcyclopropyl)propyl)octan-4-ylmethanesulfonate 89 was obtained as a colorless oil.

Synthesis of Compound 90

Using an analogous procedure to that described for the synthesis of 62,5-((1,8-bis(2-pentyl cyclopropyl)-5-(3-(2-pentylcyclopropyl)propyl)octan-4-yl)oxy)-N,N-dimethylpentan-1-amine 90 (580mg) was obtained as a pale yellow oil. Rf 0.48 (10% MeOH—CH₂Cl₂).

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. The disclosures of all articles andreferences, including patent applications, patents, PCT publications,and Genbank Accession Nos. and sequences described therein, areincorporated herein by reference for all purposes.

1-39. (canceled)
 40. A lipid particle comprising mRNA as a therapeuticagent and a lipid having a structural Formula (I):X-A-Y—Z;  (I) or salts thereof, wherein: X is alkylamino; A is C₁ to C₆optionally substituted alkyl, wherein said C₁ to C₆ optionallysubstituted alkyl can be saturated or unsaturated, and wherein A may ormay not be present; Y is selected from the group consisting of ketal,ester, optionally substituted carbamate, ether, and optionallysubstituted amide; and Z has the formula:

wherein, R₁, R₂, and R₃ are each independently selected from the groupconsisting of C₈ to C₁₁ alkyl, wherein each of R₁, R₂, and R₃ canindependently be saturated or unsaturated, and wherein each of R₁, R₂,and R₃ is optionally substituted.
 41. The lipid particle of claim 40,wherein each of the R₁, R₂, and R₃ alkyl chains has a length of from C₉to C₁₀.
 42. The lipid particle of claim 40, wherein at least one of theR₁, R₂, and R₃ alkyl chains comprises a cycloalkyl moiety and a doublebond.
 43. The lipid particle of claim 40, wherein at least one of theR₁, R₂, and R₃ alkyl chains comprises a cycloalkyl moiety or a doublebond.
 44. The lipid particle of claim 40, wherein X is selected from thegroup consisting of dimethylamino, diethylamino and ethylmethylamino.45. The lipid particle of claim 40, wherein the lipid is selected fromthe group consisting of:

or a salt thereof.
 46. The lipid particle of claim 40, wherein theparticle further comprises a non-cationic lipid.
 47. The lipid particleof claim 46, wherein the non-cationic lipid is selected from the groupconsisting of a phospholipid, cholesterol, or a mixture of aphospholipid and cholesterol.
 48. The lipid particle of claim 47,wherein the phospholipid is dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine (DSPC), or a mixture thereof.
 49. Thelipid particle of claim 40, wherein the particle further comprises aconjugated lipid that inhibits aggregation of particles.
 50. The lipidparticle of claim 49, wherein the conjugated lipid that inhibitsaggregation of particles is a polyethyleneglycol (PEG)-lipid conjugate.51. The lipid particle of claim 40, wherein when A, R₁, R₂, and R₃ areeach independently optionally substituted, at least one hydrogen atom isreplaced with a substituent selected from oxo, halogen, heterocycle,—CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y),—C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO—R^(x), and—SO—NR^(x)R^(y), wherein n is 0, 1, or 2, R^(x) and R^(y) are the sameor different and are independently hydrogen, alkyl, or heterocycle, andeach of the alkyl and heterocycle substituents may be furthersubstituted with one or more of oxo, halogen, —OH, —CN, alkyl, —OR^(x),heterocycle, —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y),—C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x), and—SO_(n)NR^(x)R^(y); and wherein when Y is independently optionallysubstituted, Y is independently optionally substituted with a saturatedor unsaturated alkyl group.
 52. The lipid particle of claim 40, whereinA, Y, R₁, R₂, and R₃ are not substituted.